Method for making polynucleotides having desired characteristics

ABSTRACT

In silico nucleic acid recombination methods, related integrated systems utilizing genetic operators and libraries made by in silico shuffling methods are provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 11/339,090 filedJan. 24, 2006 now U.S. Pat. No. 7,620,502, which is a continuation of“METHODS FOR MAKING CHARACTER STRINGS, POLYNUCLEOTIDES AND POLYPEPTIDESHAVING DESIRED CHARACTERISTICS” by Selifonov et al., U.S. Ser. No.09/618,579 filed Jul. 18, 2000 now U.S. Pat. No. 7,024,312, which is acontinuation-in-part of “METHODS FOR MAKING CHARACTER STRINGS,POLYNUCLEOTIDES AND POLYPEPTIDES HAVING DESIRED CHARACTERISTICS” bySelifonov et al., U.S. Ser. No. 09/539,486 filed Mar. 30, 2000 now U.S.Pat. No. 7,058,515, which is a continuation-in-part of “METHODS FORMAKING CHARACTER STRINGS, POLYNUCLEOTIDES AND POLYPEPTIDES HAVINGDESIRED CHARACTERISTICS” by Selifonov et al., filed Jan. 18, 2000, U.S.Ser. No. 09/494,282 now U.S. Pat. No. 6,917,882, and “METHODS FOR MAKINGCHARACTER STRINGS, POLYNUCLEOTIDES AND POLYPEPTIDES HAVING DESIREDCHARACTERISTICS” by Selifonov et al., filed Jan. 18, 2000, U.S. Ser. No.PCT/US00/01202; which are continuation-in-part applications of “METHODSFOR MAKING CHARACTER STRINGS, POLYNUCLEOTIDES AND POLYPEPTIDES HAVINGDESIRED CHARACTERISTICS” by Selifonov et al., U.S. Ser. No. 09/416,375,filed Oct. 12, 1999 now abandoned, which is a non provisional of“METHODS FOR MAKING CHARACTER STRINGS, POLYNUCLEOTIDES AND POLYPEPTIDESHAVING DESIRED CHARACTERISTICS” by Selifonov and Stemmer, U.S. Ser. No.60/116,447, filed Jan. 19, 1999 and a non-provisional of “METHODS FORMAKING CHARACTER STRINGS, POLYNUCLEOTIDES AND POLYPEPTIDES HAVINGDESIRED CHARACTERISTICS” by Selifonov and Stemmer, U.S. Ser. No.60/118,854, filed Feb. 5, 1999.

U.S. Ser. No. 09/618,579 is also a continuation-in-part of“OLIGONUCLEOTIDE MEDIATED NUCLEIC ACID RECOMBINATION” by Crameri et al.,Filed Jan. 18, 2000, U.S. Ser. No. 09/484,850 now U.S. Pat. No.6,368,861 and of “OLIGONUCLEOTIDE MEDIATED NUCLEIC ACID RECOMBINATION”by Crameri et al., Filed Jan. 18, 2000, U.S. Ser. No. PCT/US00/01203,which are continuation-in-part applications of “OLIGONUCLEOTIDE MEDIATEDNUCLEIC ACID RECOMBINATION” by Crameri et al., U.S. Ser. No. 09/408,392,Filed Sep. 28, 1999 now U.S. Pat. No. 6,376,246, which is anon-provisional of “OLIGONUCLEOTIDE MEDIATED NUCLEIC ACID RECOMBINATION”by Crameri et al., U.S. Ser. No. 60/118,813, filed Feb. 5, 1999 and anon-provisional of “OLIGONUCLEOTIDE MEDIATED NUCLEIC ACID RECOMBINATION”by Crameri et al., U.S. Ser. No. 60/141,049, filed Jun. 24, 1999.

U.S. Ser. No. 09/618,579 is also a continuation-in-part of “METHODS OFPOPULATING DATA STRUCTURES FOR USE IN EVOLUTIONARY SIMULATIONS” bySelifonov and Stemmer, U.S. Ser. No. PCT/US00/01138, filed Jan. 18, 2000which is a continuation-in-part of “METHODS OF POPULATING DATASTRUCTURES FOR USE IN EVOLUTIONARY SIMULATIONS” by Selifonov andStemmer, U.S. Ser. No. 09/416,837, filed Oct. 12, 1999 now abandoned.

U.S. Ser. No. 09/618,579 is also related to “USE OF CODON VARIEDOLIGONUCLEOTIDE SYNTHESIS FOR SYNTHETIC SHUFFLING” by Welch et al., U.S.Ser. No. 09/408,393, filed Sep. 28, 1999.

The present application claims priority to and benefit of each of theapplications listed in this section, as provided for under 35 U.S.C.§119(e) and/or 35 U.S.C. §120, as well as any other applicable statue orrule, as appropriate. All of the preceding applications are incorporatedherein by reference.

COPYRIGHT NOTIFICATION

Pursuant to 37 C.F.R. 1.71 (e), Applicants note that a portion of thisdisclosure contains material which is subject to copyright protection.The copyright owner has no objection to the facsimile reproduction byanyone of the patent document or patent disclosure, as it appears in thePatent and Trademark Office patent file or records, but otherwisereserves all copyright rights whatsoever.

FIELD OF THE INVENTION

This invention is in the field of genetic algorithms and the applicationof genetic algorithms to nucleic acid shuffling methods.

BACKGROUND OF THE INVENTION

Recursive nucleic acid recombination (“shuffling”) provides for therapid evolution of nucleic acids, in vitro and in vivo. This rapidevolution provides for the generation of encoded molecules (e.g.,nucleic acids and proteins) with new and/or improved properties.Proteins and nucleic acids of industrial, agricultural and therapeuticimportance can be created or improved through DNA shuffling procedures.

A number of publications by the inventors and their co-workers describeDNA shuffling. For example, Stemmer et al. (1994) “Rapid Evolution of aProtein” Nature 370:389-391; Stemmer (1994) “DNA Shuffling by RandomFragmentation and Reassembly: in vitro Recombination for MolecularEvolution,” Proc. Natl. Acad. USA 91:10747-10751; Stemmer, U.S. Pat. No.5,603,793 “METHODS FOR IN VITRO RECOMBINATION;” Stemmer et al., U.S.Pat. No. 5,830,721, “DNA MUTAGENESIS BY RANDOM FRAGMENTATION ANDREASSEMBLY;” and Stemmer et al., U.S. Pat. No. 5,811,238 “METHODS FORGENERATING POLYNUCLEOTIDES HAVING DESIRED CHARACTERISTICS BY ITERATIVESELECTION AND RECOMBINATION” describe, e.g., a variety of shufflingtechniques.

Many applications of DNA shuffling technology have also been developedby the inventors and their co-workers. In addition to the publicationsnoted above, Minshull et al., U.S. Pat. No. 5,837,458 METHODS ANDCOMPOSITIONS FOR CELLULAR AND METABOLIC ENGINEERING provides for theevolution of new metabolic pathways and the enhancement ofbio-processing through recursive shuffling techniques. Crameri et al.(1996), “Construction And Evolution Of Antibody-Phage Libraries By DNAShuffling” Nature Medicine 2(1):100-103 describe, e.g., antibodyshuffling for antibody phage libraries. Additional details regarding DNAshuffling can also be found in various published applications, such asWO95/22625, WO97/20078, WO96/33207, WO97/33957, WO98/27230, WO97/35966,WO98/31837, WO98/13487, WO98/13485 and WO989/42832.

A number of the publications of the inventors and their co-workers, aswell as other investigators in the art also describe techniques whichfacilitate DNA shuffling, e.g., by providing for reassembly of genesfrom small fragments of genes, or even oligonucleotides encoding genefragments. In addition to the publications noted above, Stemmer et al.(1998) U.S. Pat. No. 5,834,252 “END COMPLEMENTARY POLYMERASE REACTION”describe processes for amplifying and detecting a target sequence (e.g.,in a mixture of nucleic acids), as well as for assembling largepolynucleotides from fragments.

Review of the foregoing publications reveals that DNA shuffling is animportant new technique with many practical applications. Thus, newtechniques which facilitate DNA shuffling are highly desirable. Inparticular, techniques which reduce the number of physical manipulationsneeded for shuffling procedures would be particularly useful. Thepresent invention provides significant new DNA shuffling protocols, aswell as other features which will be apparent upon complete review ofthis disclosure.

SUMMARY OF THE INVENTION

The present invention provides new “in silico” DNA shuffling techniques,in which part, or all, of a DNA shuffling procedure is performed ormodeled in a computer system, avoiding (partly or entirely) the need forphysical manipulation of nucleic acids. These approaches arecollectively termed Genetic Algorithm Guided Gene Synthesis or “GAGGS.”

In a first aspect, the invention provides methods for obtaining a“chimeric” or “recombinant” polynucleotide or polypeptide (or otherbio-polymer) having a desired characteristic. In the methods, at leasttwo parental character strings encoding sequence information for one ormore polypeptides and/or for one or more single-stranded ordouble-stranded polynucleotides are provided. All or a part of thesequences (i.e., one or more subsequence regions) contain areas ofidentity and areas of heterology. A set of character strings of apre-defined or selected length is provided that encodes single-strandedoligonucleotide sequences which include overlapping sequence fragmentsof at least a part of each of the parental character strings, and/or atleast a part of polynucleotide strands complementary to the parentalcharacter strings.

In one class of embodiments, the invention provides methods ofgenerating libraries of biological polymers. The method includegenerating a diverse population of character strings in a computer,where the character strings are generated by alteration (recombination,mutagenesis, etc.) of pre-existing character strings. The diversepopulation of character strings is then synthesized to comprise thelibrary of biological polymers (nucleic acids, polypeptides, peptidenucleic acids, etc.). Typically, the members of the library ofbiological polymers are selected for one or more activity. In onerecursive aspect of the invention, an additional library or anadditional set of character strings is filtered by subtracting theadditional library or the additional set of character strings withmembers of the library of biological polymers which display activitybelow a desired threshold. In an additional or complementary recursiveaspect of the invention, the additional library or additional set ofcharacter strings is filtered by biasing the additional library, or theadditional set of character strings, with members of the library ofbiological polymers which display activity above a desired threshold.

A set of single-stranded oligonucleotides made according to the set ofsequences defined in the character strings is provided. Part or all ofthe single stranded nucleotides produced are pooled under denaturing orannealing conditions, where at least two single-strandedoligonucleotides represent parts of two different parental sequences.The resultant population of the single-stranded oligonucleotides isincubated with a polymerase under conditions which result in annealingof the single-stranded fragments at areas of identity to form pairs ofannealed fragments. These areas of identity are sufficient for onemember of the pair to prime replication of the other, resulting in anincrease in the length of the oligonucleotides. The resulting mixture ofdouble- and single-stranded oligonucleotides are denatured intosingle-stranded fragments. These steps are repeated, such that at leasta part of the resultant mixture of single-stranded chimeric andmutagenized polynucleotides are used in the steps of subsequent cycles.Recombinant polynucleotides having evolved toward a desired property areselected or screened for.

In another aspect, the invention provides for the use of geneticoperators, e.g., in a computer. In these methods, sequence stringscorresponding to the oligonucleotides noted above are selected by thecomputer from sequence strings corresponding to one or more of thefollowing sets of single-stranded oligonucleotides:

a) oligonucleotides synthesized to contain randomly or non-randomlypre-selected mutations of the parental sequences according to modifiedsequences including replacement of one or more characters with anothercharacter, or deletion or insertion of one or more characters;

b) oligonucleotide sequences synthesized to contain degenerate, mixed orunnatural nucleotides, at one or more randomly or non-randomlypre-selected positions; and,

c) chimeric oligonucleotides synthesized according to artificialsequences of character substrings designed to contain joined partialsequences of at least two parental sequences.

In certain embodiments, oligonucleotides of set (c) contain one or moremutated or degenerate positions defined in sets (a) and (b). Theoligonucleotides of set (c) are optionally chimeric nucleotides withcrossover points selected according to a method allowing identificationof a plurality of character substrings displaying pairwise identity(homology) between any or all of the string pairs comprising sequencesof different parental character strings.

Crossover points for making chimeric oligonucleotide sequences areoptionally selected randomly, or approximately in the middle of each ora part of the identified pairwise identity (homology) areas, or by anyother set of selection criteria.

In one aspect, at least one crossover point for at least one chimericoligonucleotide sequence is selected from those not within detectedidentity areas.

In one aspect, the mixtures of single stranded oligonucleotidesdescribed above are pooled at least once with an additional set ofpolynucleotides comprising one or more double-stranded orsingle-stranded polynucleotide encoded by a part and/or by an entirecharacter string of any of the parental sequences provided, and/or byanother character string(s) which contains areas of identity and areasof heterology with any of the parental character strings provided.

The polynucleotides from the additional set of polynucleotides can beobtained by oligonucleotide synthesis of oligonucleotides correspondingto any parental character string (or homolog thereof), or by randomfragmentation (e.g., by enzymatic cleavage e.g., by a DNAse, or bychemical cleavage of the polynucleotide) and/or by a restriction-enzymefragmentation of polynucleotides encoded by character strings definedabove, and/or by another character string(s) which contains areas ofidentity and areas of heterology with any of the parental characterstrings provided. That is, any nucleic acid generated by GAGGS can befurther modified by any available method to produce additionallydiversified nucleic acids. Furthermore, any diversified nucleic acid canserve as a substrate for further rounds of GAGGS.

The above methods are suitably adapted to a wide range of lengths forsynthetic oligos (e.g., 10-20 nucleotides or more, 20-40 nucleotides ormore, 40-60 nucleotides or more, 60-100 nucleotides or more, 100-150nucleotides or more, etc.), a wide variety of types of parentalsequences (e.g., for therapeutic proteins such as EPO, insulin, growthhormones, antibodies or the like; agricultural proteins such as planthormones, disease resistance factors, herbicide resistance factors(e.g., p450s) industrial proteins (e.g., those involved in bacterial oildesulfurization, synthesis of polymers, detoxification proteins andcomplexes, fermentation or the like)) and for a wide variety in thenumber of selection/screening cycles (e.g., 1 or more cycle, 2 or morecycle, 3-4 or more cycles, 10 or more cycles, 10-50 or more cycles,50-100 or more cycles, or more than 100 cycles). Rounds of GAGGSevolution can be alternated with rounds of physical nucleic acidshuffling and/or selection assays under various formats (in vivo, or invitro). Selected nucleic acids (i.e., those with desirable properties)can be deconvoluted by sequencing or other procedures such asrestriction enzyme analysis, real-time PCR analysis or the like, so thatthe processes can be started over using the sequence information toguide gene synthesis, e.g., without any physical manipulation of DNAobtained from previous GAGGS rounds.

Typically in the methods above, synthesis of polynucleotides fromsingle-stranded oligonucleotides is performed by assembly PCR. Otheroptions for making nucleic acids include ligation reactions, cloning andthe like.

In typical embodiments, the sets of character strings, encodingsingle-strand oligonucleotides comprising fragments of parental strings,including chimeric and mutated/degenerate fragments of a pre-definedlength, are generated using a device comprising a processing element,such as a computer with software for sequence string manipulation.

In one aspect, the invention provides for single parent GAGGS. Thesemethods are set forth in more detail in the examples herein.

The invention also provides methods of producing recombinant nucleicacids using bridging oligonucleotide selection strategies. In themethods, two or more parental nucleic acid sequences are provided.Cross-over sites are selected for recombination between the two or moreparental nucleic acid sequences, thereby defining one or morerecombinant nucleic acids that result from a cross-over between at leasttwo of the two or more parental nucleic acids. Corresponding bridgingoligonucleotides are defined. A recombinant sequence for at least one ofthe one or more recombinant nucleic acids is determined. The at leastone recombinant sequence is selected in silico for one or more expectedactivity and the at least one recombinant sequence is synthesized. Thesynthetic step is typically performed by providing fragments of the twoor more parental nucleic acids and at least one of corresponding bridgeoligonucleotides, hybridizing the fragments and the bridgeoligonucleotides and elongating the hybridized fragments with apolymerase or a ligase. This dramatically simplifies the overallsynthesis strategy for creating recombinant nucleic acids between two ormore parental sequences, including sequences which display low levels ofsequence similarity.

The present invention further provides methods of producing one or morerecombinant nucleic acids or encoded polypeptides. In the methods, aplurality of first nucleic acid or first polypeptide sequences areprovided. The first nucleic acid or polypeptide sequences optionallycomprise homologous or non-homologous sequences, and the sequences cancomprise artificial or natural sequences. Cross-over sequences areselected between the plurality of first nucleic acid or firstpolypeptide sequences by defining structural, statistical, or logicalcriteria for the cross-over sequences in silico. 104. Defining thestructural logical or statistical criteria can include any of a varietyof methods set for the herein, including performing structural modelingof at least one of the first polypeptide sequences to define one or moreregion of structural interest in the at least one first polypeptidesequence and selecting one or more cross-over sequence to preserve ordisrupt the region of structural interest; defining a structural orsequence-based motif in at least one of the first polynucleotide orpolypeptide sequences to define one or more conserved region in the atleast one first polynucleotide or polypeptide sequence and selecting oneor more cross-over sequence to preserve or disrupt the motif:identifying one or more nucleotides or amino acids within at least oneof the first polynucleotide or polypeptide sequences which showsactivity or structural co-variance for one or more desired activities orstructural features of the first polynucleotide or polypeptide sequenceand selecting one or more cross-over sequence to preserve or disrupt theco-variance; performing an energy minimization analysis of the firstpolynucleotide or polypeptide sequence and selecting one or morecross-over sequence to preserve or disrupt energy minimization of thefirst polynucleotide or polypeptide sequence; performing a stabilityanalysis of the first polynucleotide or polypeptide sequence andselecting one or more cross-over sequence to preserve or disruptstability of the of the first polynucleotide or polypeptide sequence atleast one recombinant sequence; comparing an energy minimized model ofthe first polynucleotide or polypeptide sequence to an energy minimizedmodel of one or more parental nucleic acid from which the firstpolynucleotide or polypeptide sequence was derived and selecting one ormore cross-over sequence to preserve or alter energy minimization of thefirst polynucleotide or polypeptide sequence; performing proteinthreading on one or more first polypeptide sequence and selecting thecross-over sequences to maintain or disrupt protein threading;performing one or more of: PDA, a branch-and-terminate combinatorialoptimization analysis, a dead end elimination, a genetic or mean-fieldanalysis, or analysis of protein folding by threading, of the least oneof the first polynucleotide or polypeptide sequence, or the like.

Generally, a plurality of recombinant nucleic acids comprising orencoding the cross-over sequences are synthesized. This can include, forexample, artificially synthesizing a plurality of recombinant nucleicacids comprising or encoding the cross-over sequences comprisessynthesizing a plurality or oligonucleotides, one or more of whichencodes part or all of one or more of the cross-over sequences. Theplurality of oligonucleotides are typically incubated with a polymeraseor a ligase (or both a polymerase and a ligase).

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1C present a flow chart describing a portion of directedevolution by GAGGS.

FIG. 2 is a flow chart describing a portion of directed evolution byGAGGS. The flow chart of FIG. 2 is optionally contiguous from FIG. 1.

FIGS. 3A-3C present a flow chart describing a portion of directedevolution by GAGGS. The now chart of FIG. 3 is optionally contiguousfrom FIG. 2.

FIGS. 4A-4 D present a flow chart describing a portion of directedevolution by GAGGS. The flow chart of FIG. 4 is optionally contiguousfrom FIG. 3.

FIGS. 5A and 5B present a chart and relational tree showing percentsimilarity for different subtilisins (an exemplar shuffling target).

FIG. 6 is a pairwise dot-plot alignment showing homology areas fordifferent subtilisins.

FIG. 7 is a pairwise dot-plot alignment showing homology areas for 7different parental subtilisins.

FIG. 8, Panels A-C are pairwise histograms showing conditionsdetermining probability of crossover point selection can beindependently controlled for any region over a selected gene length, aswell as independently for the pairs of parents.

FIG. 9 is a chart showing introducing indexed crossover points markerinto the sequence of each parent.

FIG. 10 shows a procedure for oligonucleotide assembly to make nucleicacids.

FIG. 11 is a continuation of FIG. 13 showing an oligonucleotide assemblyscheme.

FIGS. 12A and 12B present a difference plot and relatedness tree forshuffling Naphthalene deoxygenase.

FIG. 13 is a schematic of a digital system of the invention.

FIGS. 14A and 14B present a schematic showing a geometric relationshipbetween nucleotides.

FIG. 15 is a schematic of an HMM matrix.

DETAILED DESCRIPTION

In the methods of the invention, “genetic” or “evolutionary” algorithmsare used to produce sequence strings which can be converted intophysical molecules, shuffled and tested for a desired property. Thisgreatly expedites forced evolution procedures, as the ability topre-select substrates for shuffling reduces actual physical manipulationof nucleic acids in shuffling protocols. In addition, the use ofcharacter strings as “virtual substrates” for shuffling protocols, whencoupled with gene reconstruction methods, eliminates the need to obtainparental physical molecules encoding genes.

Genetic algorithms (GAs) are used in a wide variety of fields to solveproblems which are not fully characterized or which are too complex toallow for full characterization, but for which some analyticalevaluation is available. That is, GAs are used to solve problems whichcan be evaluated by some quantifiable measure for the relative value ofa solution (or at least the relative value of one potential solution incomparison to another). The basic concept of a genetic algorithm is toencode a potential solution to a problem as a series of parameters. Asingle set of parameter values is treated as the “genome” or geneticmaterial of an individual solution. A large population of candidatesolutions are created. These solutions can be bred with each other forone or more simulated generations under the principle of survival of thefittest, meaning the probability that an individual solution will passon some of its parameter values to subsequent solution sets is directlyrelated to the fitness of the individual (i.e., how good that solutionis relative to the others in the population for the selected parameter).Breeding takes place through use of operators such as crossovers whichsimulate basic biological recombination, and mutation. The simpleapplication of these operators with reasonable selection mechanisms hasproduced startlingly good results over a wide range of problems.

An introduction to genetic algorithms can be found in David E. Goldberg(1989) Genetic Algorithms in Search, Optimization and Machine Learning,Addison-Wesley Pub Co; ISBN: 0201157675 and in Timothy Masters (1993)Practical Neural Network Recipes in C++ (Book&Disk edition) Academic Pr;ISBN: 0124790402. A variety of more recent references discuss the use ofgenetic algorithms used to solve a variety of difficult problems. See,e.g., garage.cse.msu.edu/papers/papers-index.html (on the world wideweb) and the references cited therein; gaslab.cs.unr.edu/ (on the worldwide web) and the references cited therein; aic.nrl.navy.mil/ (on theworld wide web) and the references cited therein;cs.gmu.edu/research/gag/ (on the world wide web) and the referencescited therein and cs.gmu.edu/research/gag/pubs.html (on the worldwideweb) and the references cited therein.

In the present invention, a genetic algorithm (GA) is used to provide acharacter string-based representation of the process of generatingbiopolymer diversity (computational evolution of character strings byapplication of one or more genetic operators to a provided population(e.g., a parent library) of character strings, e.g. gene sequences).

A representation of a GA-generated character string population (or“derivative library”) is used as a sequence instruction set in a formsuitable to control polynucleotide synthesis (e.g. via non-error-pronesynthesis, error-prone synthesis, parallel synthesis, pooled synthesis,chemical synthesis, chemoenzymatic synthesis, (including assembly PCR ofsynthetic oligonucleotides), and the like). Synthesis of polynucleotidesis conducted with sequences encoded by a character string in thederivative library. This creates a physical representation (a library ofpolynucleotides) of the computation-generated “gene” (or any othercharacter string) diversity.

Physical selection of the polynucleotides having desired characteristicsis also optionally (and typically) conducted. Such selection is based onresults of physical assays of properties of polynucleotides, orpolypeptides, whether translated in-vitro, or expressed in-vivo.

Sequences of those polynucleotides found to have desired characteristicsare deconvoluted (e.g., sequenced, or, when positional information isavailable, by noting the position of the polynucleotide). This isperformed by DNA sequencing, by reading a position on an array, realtime PCR (e.g., TAQMAN™ PCR Reagents), restriction enzyme digestion, orany other method noted herein, or currently available.

These steps are optionally repeated, e.g., for 1-4 or more cycles, eachtime optionally using the deconvoluted sequences as an informationsource to generate a new, modified set of character strings to start theprocedure with. Of course, any nucleic acid which is generated in silicocan be synthesized and shuffled by any known DNA shuffling method,including those taught in the references by the inventors and theirco-workers cited herein. Such synthesized DNAs can also be mutagenizedor otherwise modified according to existing techniques.

In summary, GAGGS is an evolutionary process which includes aninformation manipulation step (application of a genetic algorithm to acharacter string representing a biopolymer such as a nucleic acid orprotein), to create a set of defined information elements (e.g.,character strings) which serve as templates for synthesizing physicalnucleic acids. The information elements can be placed into a database orotherwise manipulated in silico, e.g., by the recursive application of aGA to the sequences which are produced. Corresponding physical nucleicacids can be subjected to recombination/selection or other diversitygenerating procedures, with the nucleic acids being deconvoluted (e.g.,sequenced or otherwise analyzed) and the overall process repeated, asappropriate, to achieve a desired nucleic acid.

Example Advantages of GAGGS

There are a variety of advantages to GAGGS as compared to the prior art.For example, physical access to genes/organisms is not required forGAGGS, as sequence information is used for oligo design and selection. Avariety of public databases provide extensive sequence information,including, e.g., Genbank™ and those noted supra. Additional sequencedatabases are available on a contract basis from a variety of companiesspecializing in genomic information generation and storage.

Similarly, sequences from inaccessible, non-cultivable organisms can beused for GAGGS. For example, sequences from pathogenic organisms can beused without actual handling of the pathogens. All of the sequence typessuitable for physical DNA shuffling, including damaged and incompletegenes (e.g., pseudo genes), are amenable to GAGGS.

All genetic operators, including different types of mutagenesis andcrossovers can be fully and independently controlled in a reproduciblefashion, removing human error and variability from physical experimentswith DNA manipulations. GAGGS has applicability to the self-learningcapability of artificial intelligence (optimization algorithm outputparameter profiles based on feedback entry of yields, success rates andfailures of physical screens, etc.).

In GAGGS procedures, sequences with frame-shift mutations (which aregenerally undesirable) are eliminated or fixed (discarded from thecharacter set, or repaired, in silico). Similarly, entries withpremature terminations are discarded or repaired and entries with lossof sequence features known to be important for display of a desiredproperty (e.g. conservative ligands for metal binding) are discarded orrepaired.

Furthermore, wild-type parents do not contaminate derivative librarieswith multiple redundant parental molecules, as, in one preferredembodiment, only a priori modified genes are subjected to physicalshuffling and/or screening (which, in some cases can be expensive, orlow throughput, or otherwise less than ideal, depending on the assayavailable).

In addition, because no actual physical recombination is required,protein sequences can be shuffled in the same way in silico as nucleicacid sequences, and retrotranslation of the resulting shuffled sequencescan be used to alleviate codon usage problems and to minimize the numberof oligos needed to build one or more library of coding nucleic acids.In this regard, protein sequences can be shuffled in silico usinggenetic operators that are based on recognition of structural domainsand folding motifs, rather than being bound by annealing-based homologycriteria of DNA sequences, or simple homology of AA sequences.Furthermore, rational structure-based biases are easily incorporated inlibrary construction, when such information is available.

The only significant operational costs of running GAGGS is the cost ofsynthesis of large libraries of genes represented in silico. Syntheticassembly of genes can be done, e.g., by assembly PCR from 40-60 bpoligos, which can be synthesized inexpensively by current techniques.

Directed Evolution by GAGGS:

All changes in any DNA sequence during any evolutionary process can bedescribed by a finite number of events, each resulting from action of anelementary genetic operator. In any given parental sequence subspace,these changes can accurately be accounted for and simulated in aphysical representation of an evolutionary process aimed to generatesequence diversity for subsequent physical screening for desiredcharacteristics. Physical double stranded polynucleotides are notrequired for starting GAGGS processes; instead, they are generatedfollowing initial GAGGS processes with the purpose of physical screeningand/or selection, and/or as a result of this screening or selection.Generating very large libraries for screening/selection is not required.

Genetic Algorithms (GA).

CHARACTER STRINGS: in general, a character string can be anyrepresentation of an array of characters (e.g., a linear array ofcharacters provides “words” while a non-linear array can be used as acode to generate a linear array of characters). For practicing GAGGS,character strings are preferably those which encode polynucleotide orpolypeptide strings, directly or indirectly, including any encryptedstrings, or images, or arrangements of objects which can be transformedunambiguously to character strings representing sequences of monomers ormultimers in polynucleotides, polypeptides or the like (whether made ofnatural or artificial monomers).

GENETIC ALGORITHM: Genetic algorithms generally are processes whichmimic evolutionary processes. Genetic algorithms (GAs) are used in awide variety of fields to solve problems which are not fullycharacterized or too complex to allow full characterization, but forwhich some analytical evaluation is available. That is, GAs are used tosolve problems which can be evaluated by some quantifiable measure forthe relative value of a solution (or at least the relative value of onepotential solution in comparison to another). In the context of thepresent invention, a genetic algorithm is a process for selecting ormanipulating character strings in a computer, typically where thecharacter string can be corresponded to one or more biological polymer(e.g., a nucleic acid, protein, PNA, or the like). A biological polymeris any polymer which shares some structural features with naturallyoccurring polymers such as an RNAs, DNAs and polypeptides, including,e.g., RNAs, RNA analogues, DNAs, DNA analogues, polypeptides,polypeptide analogues, peptide nucleic acids, etc.

Directed Evolution of Character Strings or Objects:

A process of artificially changing a character string by artificialselection, i.e., which occurs in a reproductive population in whichthere are (1) varieties of individuals, with some varieties being (2)heritable, of which some varieties (3) differ in fitness (reproductivesuccess determined by outcome of selection for a predetermined property(desired characteristic). The reproductive population can be, e.g., aphysical population or a virtual population in a computer system.

GENETIC OPERATORS (GOs): user-defined operations, or sets of operations,each comprising a set of logical instructions for manipulations ofcharacter strings. Genetic operators are applied to cause changes inpopulations of individuals in order to find interesting (useful) regionsof the search space (populations of individuals with predetermineddesired properties) by predetermined means of selection. Predetermined(or partially predetermined) means of selection include computationaltools (operators comprising logical steps guided by analysis ofinformation describing libraries of character strings), and physicaltools for analysis of physical properties of physical objects, which canbe built (synthesized) from matter with the purpose of physicallycreating a representation of information describing libraries ofcharacter strings. In a preferred embodiment, some or all of the logicaloperations are performed in a computer.

Genetic Operators

All changes in any population of any type of character strings (and thusin any physical properties of physical objects encoded by such strings)can be described as a result of random and/or predetermined applicationof a finite set of logical algebraic functions comprising various typesof genetic operators.

In its mathematical nature, this statement is not a postulated abstractaxiom. In fact, this statement is a derivative theorem with stringentformal proof readily derived from Wiles' proof of Fermat's last theorem.The fundamental implication of the Wiles' Proof for evolutionarymolecular biology is in the proof of the central conjecture stating thatall elliptic curves are in essence modular forms. Particularly, all ofthe diversity and evolution of living matter in the universe (i.e., theplurality of objects whose properties can be described by a finitenumber of elliptical curves) can be described in the language of fivebasic arithmetic operations: addition, subtraction, multiplication,division and modular forms (i.e., evolution of life can be effectivelydescribed by a finite combination of simple changes of information in afinite population of character strings, e.g. all DNA in the universe).This being the case, it is possible to determine the language of nucleicacid-based forms of life, and to define all basic types of geneticoperators which apply to nucleic acids under evolutionary selection.

Mathematical modeling of certain genetic operations have been proposed,e.g., in Sun (1999) “Modeling DNA Shuffling” Journal of ComputationalBiology 6(1):77-90; Kelly et al. (1994) “A test of the Markovian modelof DNA evolution” Biometrics 50(3):653-64; Boehnke et al. (1991)“Statistical methods for multipoint radiation hybrid mapping” Am. J.Hum. Genet. 49:1174-1188; Irvine et al. (1991) “SELEXION: systematicevolution of ligands by exponential enrichment with integratedoptimization by non-linear analysis” J. Mol. Biol. 222:739-761; Landerand Waterman (1988) Genomic mapping by fingerprinting random clones: amathematical analysis” Genomics 2:231-239; Lange (1997) Mathematical andStatistical Methods for Genetic Analysis Springer Verlag, NY: Sun andWaterman (1996) “A mathematical analysis of in vitro molecularselection-amplification” J. Mol. Biol. 258:650-660; Waterman (1995)Introduction to Computational Biology Chapman and Hall, London, UK.

The following provides a description of certain basic genetic operationsapplicable to the present invention.

MULTIPLICATION (including duplication and replication) is a form ofreproduction of character strings, producing additional copies ofcharacter strings comprising parental population/library of strings.Multiplication operators can have many variations. They can be appliedto individual strings or to groups of identical or non-identicalstrings. Selecting groups of strings for multiplication can be random orbiased.

MUTATION: all mutation types in each member of a set of strings can bedescribed by several simple operations which can be reduced to elementscomprising replacement of one set of the characters with another set ofcharacters. One or more characters can be mutated in a single operation.When more than one character is mutated, the set of characters may ormay not be continuous over an entire string length (a feature useful tosimulate closely clustered mutations by certain chemical mutagens). ASingle point mutation operator replaces a single character with anothersingle character. The nature of the new characters can vary, and theycan be from the same set of characters making up parental strings, orfrom different, (e.g., to represent degenerate nucleobases, unnaturalnucleobases or amino acids, etc). A Deletion mutation is a more complexoperator which removes one or more characters from strings. Individualsingle point deletions in nucleic acid-encoding strings may be notdesirable for manipulating strings representing polynucleotidesequences; however, 3× clustered (continuous or dispersed) deletions maybe acceptable (“triple deletion frameshifts”). Single point deletions,though, are useful and acceptable for evolutionary computation ofstrings encoding polypeptides. Insertion mutations are operationallysimilar to deletions, except that one or more new characters areinserted. The nature of the added characters optionally vary, and theycan be from the same set of characters making up parental strings, orfrom different, (e.g., to represent degenerate nucleobases, unnaturalnucleobases or amino acids, etc). Death can simply be defined as avariation of the deletion operator. It takes place when the result of anapplication of a genetic operator (or combinations thereof) yields adeletion of an entire individual character string, or entire (sub)population of character strings. Death can also be defined as avariation of an elitism-prone multiplication operator (multiplication ofvalues defining abundance level of one or more strings by zero). Deathcan also be defined as a default non-selection action in operators,effecting selections of sub-populations of string and transfermanipulations with various sorting and indexing operations of indexedlibraries of strings (all non-transferred strings can be considered asdead or non-existent for subsequent computations).

FRAGMENTATION OF STRINGS are an important class of non-elemental(complex) optional operators which can have advantages for simulatingevolution of strings in various formats of DNA shuffling. Operationally,fragmentation can be described as a formal variation of a combination ofa deletion operator and a multiplication operator. One of skill willappreciate, however, that there are many other simple algorithmicoperations which allow any given character string be fragmented to givea progeny of shorter strings. Fragmentation operations may be random orbiased. Different ranges of fragment sizes can be predetermined. Stringfragments may be left in the same population with parental stings, orthey may be transferred to different population. Strings fragments fromvarious population strings can be pooled to from new populations.

CROSSOVER (RECOMBINATION)—This operator formally comprises joining acontinuous part of one string with a continuous part of another stringin such a way that one or two hybrid strings are formed (chimeras),where each of the chimeras contain at least two connected continuousstring areas each comprising partial sequence of two differentrecombining strings. The area/point where sequence characters fromdifferent parental strings, is termed the crossover/recombinationarea/point. Crossover operations can be combined with mutationoperations affecting one or more characters of the recombined strings ina proximity of the crossover area/point of joining. When appliedrecursively to a population of character strings, complex chimerascomprising consequently connected partial sequences of more than twoparental strings can be formed.

LIGATION is a variation of an insertion mutation operator whereessentially the entire content of one string is combined with the entirecontent of another string in a way that the last character of one stringis followed by the first character of another string. Ligation operationcan be combined with mutation operation affecting one or more charactersof the ligated strings in a proximity of the point of joining. Ligationcan also be viewed as a means of forming chimeras.

ELITISM is a concept that provides a useful form of bias which imposesdiscriminating criteria for use of any of the genetic operators, andvarious types of positive and negative biases can be designed andimplemented. The rational for the design of elitist operators is basedon the concept of fitness. Fitness can be determined using stringanalysis tools which recognize various sequence-specific features(GC-content, frameshifts, terminations, sequence length, specificsubstrings, homology properties, ligand-binding and folding motifs, etc)and/or indexed correlated parameters acquired from physical selection ofphysical representations of character strings (enzyme activity,stability, ligand binding, etc.). It is understood that differentelitism criteria can be applied separately to any of the above describedgenetic operators, or combinations of operators. It is also possible touse elitism, in the same evolutionary computation process, with severaloperators of the same type, where input/output parameters of each of thesimilar operators can be controlled independently (or interdependently).Different elitism criteria can be used to control changes in the stringcharacter populations caused by action of each of the individualoperators.

SEQUENCE HOMOLOGY or SEQUENCE SIMILARITY is an especially important formof sequence-specific elitism useful for controlling changes inpopulations of character strings caused by crossover/recombinationoperators in those genetic algorithms used to evolve character stringsencoding polynucleotide and polypeptide sequences.

Various approaches, methods and algorithms known in the art can be usedto detect homology or similarity between different character strings.Optimal alignment of sequences for comparison can be conducted, e.g., bythe local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482(1981), by the homology alignment algorithm of Needleman & Wunsch, J.Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson& Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerizedimplementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA inthe Wisconsin Genetics Software Package, Genetics Computer Group, 575Science Dr., Madison, Wis.), or by even by visual inspection (seegenerally, Ausubel et al., infra).

One example algorithm that is suitable for determining percent sequenceidentity and sequence similarity is the BLAST algorithm, which isdescribed in Altschul et al., J. Mol. Biol. 215:403-410 (1990). Softwarefor performing BLAST analyses is publicly available through the NationalCenter for Biotechnology Information (ncbi.nlm.nih.gov/ on the worldwide web). This algorithm involves first identifying high scoringsequence pairs (HSPs) by identifying short words of length W in thequery sequence, which either match or satisfy some positive-valuedthreshold score T when aligned with a word of the same length in adatabase sequence. T is referred to as the neighborhood word scorethreshold (Altschul et al., supra). These initial neighborhood word hitsact as seeds for initiating searches to find longer HSPs containingthem. The word hits are then extended in both directions along eachsequence for as far as the cumulative alignment score can be increased.Cumulative scores are calculated using, for nucleotide sequences, theparameters M (reward score for a pair of matching residues; always >0)and N (penalty score for mismatching residues; always <0). For aminoacid sequences, a scoring matrix is used to calculate the cumulativescore. Extension of the word hits in each direction are halted when: thecumulative alignment score falls off by the quantity X from its maximumachieved value; the cumulative score goes to zero or below, due to theaccumulation of one or more negative-scoring residue alignments; or theend of either sequence is reached. The BLAST algorithm parameters W, T,and X determine the sensitivity and speed of the alignment. The BLASTNprogram (for nucleotide sequences) uses as defaults a wordlength (W) of11, an expectation (E) of 10, a cutoff of 100, M=5, N=−4, and acomparison of both strands. For amino acid sequences, the BLASTP programuses as defaults a wordlength (W) of 3, an expectation (E) of 10, andthe BLOSUM62 scoring matrix (see Henikoff & Henikoff (1989) Proc. Natl.Acad. Sci. USA 89:10915).

In addition to calculating percent sequence identity, the BLASTalgorithm also performs a statistical analysis of the similarity betweentwo sequences (see, e.g., Karlin & Altschul (1993) Proc. Nat'l. Acad.Sci. USA 90:5873-5787). One measure of similarity provided by the BLASTalgorithm is the smallest sum probability (P(N)), which provides anindication of the probability by which a match between two nucleotide oramino acid sequences would occur by chance. For example, a nucleic acidis considered similar to a reference sequence (and, therefore,homologous) if the smallest sum probability in a comparison of the testnucleic acid to the reference nucleic acid is less than about 0.1, orless than about 0.01, and or even less than about 0.001.

An additional example of a useful sequence alignment algorithm isPILEUP. PILEUP creates a multiple sequence alignment from a group ofrelated sequences using progressive, pairwise alignments. It can alsoplot a tree showing the clustering relationships used to create thealignment. PILEUP uses a simplification of the progressive alignmentmethod of Feng & Doolittle, J. Mol. Evol. 35:351-360 (1987). The methodused is similar to the method described by Higgins & Sharp, CABIOS5:151-153 (1989). The program can align, e.g., up to 300 sequences of amaximum length of 5,000 letters. The multiple alignment procedure beginswith the pairwise alignment of the two most similar sequences, producinga cluster of two aligned sequences. This cluster can then be aligned tothe next most related sequence or cluster of aligned sequences. Twoclusters of sequences can be aligned by a simple extension of thepairwise alignment of two individual sequences. The final alignment isachieved by a series of progressive, pairwise alignments. The programcan also be used to plot a dendrogram or tree representation ofclustering relationships. The program is run by designating specificsequences and their amino acid or nucleotide coordinates for regions ofsequence comparison.

Thus, different types of similarity of with various levels of identityand length can be detected and recognized. For example, many homologydetermination methods have been designed for comparative analysis ofsequences of biopolymers, for spell-checking in word processing, and fordata retrieval from various databases. With an understanding ofdouble-helix pair-wise complement interactions among 4 principalnucleobases in natural polynucleotides, models that simulate annealingof complementary homologous polynucleotide strings can also be used as afoundation of sequence-specific elitism useful for controlling crossoveroperators.

Homology-based elitism of crossover operators can thus be used (a) tofind suitable recombination pairs of strings in a population of strings,and/or (b) to find/predetermine particularly suitable/desiredareas/points of recombination over lengths of character strings selectedfor recombination.

Setting predetermined types and stringency of similarity/homology as acondition for crossover to occur is a form of elitism for control offormation of chimeras between representative parental character stringsof various degree of homology.

RECURSIVE USE OF GENETIC OPERATORS FOR EVOLUTION OF CHARACTER STRINGS.All of the described genetic operators can be applied in a recursivemode, and specific parameters for each application occurrence can remainthe same or can be systematically or randomly varied.

RANDOMNESS IN THE APPLICATION OF GENETIC OPERATORS FOR EVOLUTION OFCHARACTER STRINGS. Each genetic operator can be applied to randomlyselected strings and/or to randomly selected positions over one or morestring's length, with occurrence frequencies randomly selected within arange.

ARRANGEMENT OF GOS IN GAS. Order determining applications of individualGOs to product derivative libraries of character strings may bedifferent and may depend on the composition of a particular set ofindividual GOs selected for practicing various formats of GAGGS. Theorder may be linear cyclic, parallel, or a combination of the three andcan typically be represented by a graph. Many GO arrangements can beused to simulate natural sexual and mutagenic processes for generatinggenetic diversity, or artificial protocols, such as single-parent orfamily DNA shuffling. However, the purpose of GA is not in limited tosimulation of some known physical DNA manipulation methods. Its main aimis in the provision of a formal and intelligent tool, based onunderstanding natural and artificial evolution processes, for creationand optimization of evolutionary protocols of practical utility whichmay provide effective advantages over currently practiced methods.

Gene Synthesis.

The physical synthesis of genes encoded by derivative libraries ofcharacter strings, obtained by operation of genetic algorithms, is theprimary means to create a physical representation of matter that isamenable to a physical assay for a desired property or to producesubstrates that are further evolved in physical diversity generationprocedures. Thus, one aspect of the present invention relates to thesynthesis of genes with sequences selected following one or morecomputer shuffling procedure as set forth herein.

For GAGGS to be a time and resource effective technology, gene synthesistechnology is used, typically to construct libraries of genes in aconsistent manner, and in close adherence to the sequencerepresentations produced by GA manipulations. GAGGS typically uses genesynthesis methods which allow for rapid construction of libraries of10⁴-10⁹ “gene” variations. This is typically adequate forscreening/selection protocols, as larger libraries are more difficult tomake and maintain and sometimes cannot be as completely sampled by aphysical assay or selection method. For example, existing physical assaymethods in the art (including, e.g., “life-and-death” selection methods)generally allow sampling of about 10⁹ variations or less by a particularscreen of a particular library, and many assays are effectively limitedto sampling of 10⁴-10⁵ members. Thus, building several smaller librariesis a preferred method, as large libraries cannot easily be completelysampled. However, larger libraries can also be made and sampled, e.g.,using high-throughput screening methods.

Gene Synthesis Technologies

There are many methods which can be used to synthesize genes withwell-defined sequences. Solely for the purpose of clarity ofillustration, this section focuses on one of the many possible andavailable types of known methods for synthesis of genes andpolynucleotides.

Current art in polynucleotide synthesis is best represented bywell-known and mature phosphoramidite chemistry which permits effectiveoligo preparation. It is possible, but somewhat impractical, to use thischemistry for routine synthesis of oligos significantly longer than 100bp, as the quality of sequence deteriorates for longer oligos, withlonger synthetic oligos generally being purified before use. Oligos of a“typical” 40-80 bp size can be obtained routinely and directly with veryhigh purity, and without substantial sequence deterioration.

For example, oligonucleotides e.g., for use in in vitroamplification/gene reconstruction methods, for use as gene probes, or asshuffling targets (e.g., synthetic genes or gene segments) are typicallysynthesized chemically according to the solid phase phosphoramiditetriester method described by Beaucage and Caruthers (1981), TetrahedronLetts., 22(20):1859-1862, e.g., using an automated synthesizer, asdescribed in Needham-VanDevanter et al. (1984) Nucleic Acids Res.,12:6159-6168. Oligonucleotides can also be custom made and ordered froma variety of commercial sources known to persons of skill. There aremany commercial providers of oligo synthesis services, and thus this isa broadly accessible technology. Any nucleic acid can be custom orderedfrom any of a variety of commercial sources, such as The MidlandCertified Reagent Company (mcrc@oligos.com). The Great American GeneCompany (genco.com, on the world wide web), ExpressGen Inc.(expressgen.com, on the world wide web), Operon Technologies Inc.(Alameda, Calif.) and many others. Similarly, peptides and antibodiescan be custom ordered from any of a variety of sources, such asPeptidoGenic (pkim@ccnet.com), HTI Bio-products, inc. (htibio.com, onthe world wide web), BMA Biomedicals Ltd (U.K.), BioSynthesis, Inc., andmany others.

As described herein, synthetic shuffling using nucleic acid ligases orpolymerases allows great freedom of oligo design and generation ofrelevant mixtures. Further, synthetic and assembly parameters permitsconsiderable control over library design. Whether polymerase mediated orligase mediated assembly methods (or a combination thereof) are used,oligos for libraries assembled by DNA ligase are synthesized, e.g., byconventional chemistry, by split-pool synthesis, or use of trinucleotidephosphoramidites, as described herein. See also, “OLIGONUCLEOTIDEMEDIATED NUCLEIC ACID RECOMBINATION” by Crameri et al., Filed Jan. 18,2000, U.S. Ser. No. PCT/US00/01203 and “USE OF CODON VARIEDOLIGONUCLEOTIDE SYNTHESIS FOR SYNTHETIC SHUFFLING” by Welch et al., U.S.Ser. No. 09/408,393, filed Sep. 28, 1999. The oligos are then assembledinto full-length sequences of interest. Oligo mixtures can be spikedwith partial or full-length homologous sequences (e.g., single ordouble-stranded sequences) e.g., from naturally occurring, synthetic orcloned sequences, to facilitate gene reassembly methods.

In any case, polymerase mediated, ligation-mediated and combinationligation/polymerase mediated assembly methods are suitable forconstruction of individual sequences and/or synthetic libraries (seealso, “OLIGONUCLETIDE MEDIATED NUCLEIC ACID RECOMBINATION” by Crameri etal., Filed Jan. 18, 2000, U.S. Ser. No. PCT/US00/01203). For example, incertain ligation mediated methods, top and bottom strand oligos can bedesigned to be overlapping, but with the oligos for each strand beingabutting, rather than overlapping, as in typical polymerase-mediatedassembly reactions. To facilitate ligation-mediated reactions, oligosare optionally phosphorylated, e.g., with a phosphorylase or a kinaseenzyme, or by chemical addition of a phosphate during or followingoligonucleotide synthesis. Phosphorylated oligos are assembled with aDNA ligase, e.g., T₄ DNA ligase or another available DNA ligase. Eitherthermostable or thermolabile ligases can be used.

In one gene assembly embodiment, a ligation chain reaction (LCR) can beperformed to achieve assembly, e.g., where a thermostable ligase is usedfor assembly. An example of an LCR-mediated gene synthesis approach isdescribed by Au et al. (1998) “Gene Synthesis by a LCR-Based Approach:High level Production of Leptin-L54 Using Synthetic Gene in Escheriacoli” Biochemical and Biophysical Research Communications 248:200-203.The gene synthetic strategies described supra and in “OLIGONUCLEOTIDEMEDIATED NUCLEIC ACID RECOMBINATION” by Crameri et al., Filed Jan. 18,2000, U.S. Ser. No. PCT/US00/01203 are generally applicable to bothligation and polymerase-mediated synthetic methods.

Another relevant demonstration of total gene synthesis from smallfragments which is readily amenable to optimization, parallelism andhigh throughout is set forth in Dillon and Rosen (Biotechniques, 1990,9(3)298-300). Simple and rapid PCR-based assembly process of a gene froma set of partially overlapping single-strand oligonucleotides, with orwithout use of ligase, can be performed. Several groups have alsodescribed successful applications of variations the same PCR-based geneassembly approach to the synthesis of various genes of increasing size,thus demonstrating its general applicability and its combinatorialnature for synthesis of libraries of mutated genes. Useful referencesinclude Sandhu, et al. (Biotechniques, 1992, 12(1)15-16), (220 bp genefrom 3 oligos of 77-86 bp); Prodromou and Pearl (Protein Engineering,1992, 5(8)827-829 (522 bp gene, from 10 oligos of 54-86 bp); Chen et al,1994 (JACS, 1194(11):8799-8800), (779 bp gene); Hayashi et al, 1994(Biotechniques, 1994, 17:310-314) and others.

More recently Stemmer et al (Gene, 1995, 164:49-53) showed that, e.g.,PCR-based assembly methods are effectively useful to build larger genesof up to at least 2.7 kb from dozens or even hundreds of synthetic 40 bpoligos. These authors also demonstrated that, from four basic stepscomprising PCR-based gene synthesis protocols (oligo synthesis, geneassembly, gene amplification, and, optionally, cloning) the geneamplification step can be omitted, if a ‘circular’ assembly PCR is used.

A number of the publications of the inventors and their co-workers, aswell as other investigators in the art also describe techniques whichfacilitate DNA shuffling, e.g., by providing for reassembly of genesfrom small fragments, or even oligonucleotides. One aspect of thepresent invention is the ability to use family shufflingoligonucleotides and cross over oligonucleotides as recombinationtemplates/intermediates in various DNA shuffling methods.

Indeed, a number of the publications by the inventors and theirco-workers, as well as other investigators in the art also describetechniques which facilitate reassembly of genes from small fragments,including oligonucleotides. In addition to the publications noted above,Stemmer et al. (1998) U.S. Pat. No. 5,834,252 END COMPLEMENTARYPOLYMERASE REACTION describe processes for amplifying and detecting atarget sequence (e.g., in a mixture of nucleic acids), as well as forassembling large polynucleotides from fragments. Crameri et al. (1998)Nature 391: 288-291 provides basic methodologies for gene reassembly, asdoes Crameri et al. (1998) Bio techniques 18(2): 194-196.

More recently, a number of gene reassembly protocols whichsimultaneously recombine and reconstruct genes have been described inseveral applications of the inventors and their co-workers, such as“OLIGONUCLEOTIDE MEDIATED NUCLEIC ACID RECOMBINATION” by Crameri et al.,filed Feb. 5, 1999 (U.S. Ser. No. 60/118,813) and filed Jun. 24, 1999(U.S. Ser. No. 60/141,049) and filed Sep. 28, 1999 (U.S. Ser No.09/408,392) and “USE OF CODON-BASED OLIGONUCLEOTIDE SYNTHESIS FORSYNTHETIC SHUFFLING” by Welch et al., Filed Sep. 28, 1999 (U.S. Ser. No.09/408,393). In these embodiments, synthetic recombination methods areused, in which oligonucleotides corresponding to different homologuesare synthesized and reassembled in PCR or ligation reactions whichinclude oligonucleotides which correspond to more than one parentalnucleic acid, thereby generating new recombined nucleic acids.

One advantage of oligonucleotide-mediated recombination is the abilityto recombine homologous nucleic acids with low sequence similarity, oreven to recombine non-homologous nucleic acids. In these low-homologyoligonucleotide shuffling methods, one or more set of fragmented nucleicacids is recombined. e.g., with a set of crossover family diversityoligonucleotides. Each of these crossover oligonucleotides have aplurality of sequence diversity domains corresponding to a plurality ofsequence diversity domains from homologous or non-homologous nucleicacids with low sequence similarity. The fragmented oligonucleotides,which are derived by comparison to one or more homologous ornon-homologous nucleic acids, can hybridize to one or more region of thecrossover oligos, facilitating recombination. Such oligonucleotide setsare selected in silico according to the methods herein.

When recombining homologous nucleic acids, sets of overlapping familygene shuffling oligonucleotides (which are derived by comparison ofhomologous nucleic acids and synthesis of oligonucleotide fragment sets,which correspond to regions of similarity and regions of diversityderived from the comparison) are hybridized and elongated (e.g., byreassembly PCR), providing a population of recombined nucleic acids,which can be selected for a desired trait or property. Typically, theset of overlapping family shuffling gene oligonucleotides include aplurality of oligonucleotide member types which have consensus regionsubsequences derived from a plurality of homologous target nucleicacids.

Typically, family gene shuffling oligonucleotide are provided byaligning homologous nucleic acid sequences to select conserved regionsof sequence identity and regions of sequence diversity. A plurality offamily gene shuffling oligonucleotides are synthesized (serially or inparallel) which correspond to at least one region of sequence diversity.Further details regarding family shuffling is found in U.S. Ser. No.09/408,392, cited above.

Sets of fragments, or subsets of fragments used in oligonucleotideshuffling approaches can be provided by cleaving one or more homologousnucleic acids (e.g., with a DNase), or, more commonly, by synthesizing aset of oligonucleotides corresponding to a plurality of regions of atleast one nucleic acid (typically oligonucleotides corresponding to afull-length nucleic acid are provided as members of a set of nucleicacid fragments). In the shuffling procedures herein, these cleavagefragments can be used in conjunction with family gene shufflingoligonucleotides, e.g., in one or more recombination reaction to producerecombinant nucleic acids.

Gene assembly by PCR from single-strand complementary overlappingsynthetic oligos is a method of choice for practicing in GAGGS.Optimization of this method can be performed e.g., including varyingoligo length, the number of oligos in the recombination reaction, thedegree of oligonucleotide overlap, levels and nature of sequencedegeneracy, specific reaction conditions and particular polymeraseenzymes used in the reassembly, and in controlling the stringency ofgene assembly to decrease or increase the number of sequence deviationsduring gene synthesis.

The method can also be practiced in a parallel mode where each of theindividual library members, including a plurality of the genes intendedfor subsequent physical screening, are synthesized in spatiallyseparated vessels, or arrays of vessels, or in a poolwise fashion, whereall, or part, of the desired plurality of genes are synthesized in asingle vessel. Many other synthesis methods for making syntheticnucleotides are also known, and specific advantages of use of one vs.another for practicing GAGGS may be readily determined by one skilled inthe art.

Sequence Deconvolution.

Sequence deconvolution is performed on those variants of polynucleotideswhich are found to have desired properties, in order to confirm changesin corresponding character strings (i.e., corresponding to physicalsequences for biopolymers) yielding desired changes in the relevantcomposition of matter (e.g., a polynucleotide, polypeptide, or thelike).

Sequencing and other standard recombinant techniques useful for thepresent invention, including for sequence deconvolution are found, e.g.,in Berger and Kimmel, Guide to Molecular Cloning Techniques, Methods inEnzymology volume 152 Academic Press, Inc., San Diego, Calif. (Berger);Sambrook et al. Molecular Cloning—A Laboratory Manual (2nd Ed.), Vol.1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989(“Sambrook”) and Current Protocols in Molecular Biology, F. M. Ausubelet al., eds., Current Protocols, a joint venture between GreenePublishing Associates, Inc. and John Wiley & Sons, Inc., (supplementedthrough 1999) (“Ausubel”)). In addition to sequencing GAGGS products,unique restriction sites can also be used for detecting particularsequences. Sufficient information to guide one of skill throughrestriction enzyme digestion is also found in Sambrook, Berger andAusubel, id.

Methods of transducing cells, including plant and animal cells, withGAGGS generated nucleic acids, e.g., for cloning and sequencing and/orfor expression and selection of encoded molecules are generallyavailable, as are methods of expressing proteins encoded by such nucleicacids. In addition to Berger, Ausubel and Sambrook, useful generalreferences for culture of animal cells include Freshney (Culture ofAnimal Cells, a Manual of Basic Technique, third edition Wiley-Liss, NewYork (1994)) and the references cited therein, Humason (Animal TissueTechniques, fourth edition W. H. Freeman and Company (1979)) andRicciardelli, et al., In Vitro Cell Dev. Biol. 25: 1016-1024 (1989).References for plant cell cloning, culture and regeneration includePayne et al. (1992) Plant Cell and Tissue Culture in Liquid Systems JohnWiley & Sons, Inc. New York, N.Y. (Payne); and Gamborg and Phillips(eds) (1995) Plant Cell, Tissue and Organ Culture; Fundamental MethodsSpringer Lab Manual, Springer-Verlag (Berlin Heidelberg New York)(Gamborg). A variety of Cell culture media are described in Atlas andParks (eds) The Handbook of Microbiological Media (1993) CRC Press, BocaRaton, Fla. (Atlas). Additional information for plant cell culture isfound in available commercial literature such as the Life ScienceResearch Cell Culture Catalogue (1998) from Sigma-Aldrich, Inc (StLouis, Mo.) (Sigma-LSRCCC) and, e.g., the Plant Culture Catalogue andsupplement (1997) also from Sigma-Aldrich, Inc (St Louis, Mo.)(Sigma-PCCS).

In vitro amplification methods can also be used to amplify and/orsequence GAGGS generated nucleic acids, e.g., for cloning, andselection. Examples of techniques sufficient to direct persons of skillthrough typical in vitro amplification and sequencing methods, includingthe polymerase chain reaction (PCR) the ligase chain reaction (LCR),Q∃-replicase amplification and other RNA polymerase mediated techniques(e.g., NASBA) are found in Berger, Sambrook, and Ausubel, id., as wellas in Mullis et al., (1987) U.S. Pat. No. 4,683,202; PCR Protocols AGuide to Methods and Applications (Innis et al. eds) Academic Press Inc.San Diego, Calif. (1990) (Innis); Arnheim & Levinson (Oct. 1, 1990) C&EN36-47; The Journal Of NIH Research (1991) 3, 81-94; Kwoh et al. (1989)Proc. Natl. Acad. Sci. USA 86, 1173; Guatelli et al. (1990) Proc. Natl.Acad. Sci. USA 87, 1874; Lomell et al. (1989) J. Clin. Chem. 35, 1826;Landegren et al., (1988) Science 241, 1077-1080; Van Brunt (1990)Biotechnology 8, 291-294; Wu and Wallace, (1989) Gene 4, 560; Barringeret al. (1990) Gene 89, 117, and Sooknanan and Malek (1995) Biotechnology13: 563-564. Improved methods of cloning in vitro amplified nucleicacids are described in Wallace et al., U.S. Pat. No. 5,426,039. Improvedmethods of amplifying large nucleic acids by PCR are summarized in Chenget al. (1994) Nature 369: 684-685 and the references therein, in whichPCR amplicons of up to 40 kb are generated. PCR reassembly techniquesare discussed supra. One of skill will appreciate that essentially anyRNA can be converted into a double stranded DNA suitable for restrictiondigestion, PCR expansion and sequencing using reverse transcriptase anda polymerase. See, Ausbel, Sambrook and Berger, all supra.

If gene synthesis is essentially error-free (stringent), and, e.g.,performed in a parallel mode where each of the individual members oflibrary was originally synthesized in spatially separated areas orcontainers (vessels), then deconvolution is performed by referencing thepositional encoding index for each intended sequence of all librarymembers. If the synthesis is performed in a poolwise fashion (or iflibrary members were pooled during selection), then one of the manyknown polynucleotide sequencing techniques is used.

Recursive GAGGS Processes.

The recursive nature of directed evolution methods, aimed atstepwise/roundwise improvement of desired properties of polynucleotidesand polypeptides, is well understood. In directed evolution (DE) byGAGGS, one or more deconvoluted character string(s), encoding sequencesof those variants displaying certain changes in level of desiredproperties (where the level is arbitrarily defined byincrease/decrease/ratios between measures of several properties), can beused to comprise a new library of character strings for a new round ofGAGGS. Recursive GAGGS, unlike typical DNA shuffling, does not usephysical manipulation of the polynucleotides in order to producesubsequent generations of gene diversity. Instead, GAGGS simply usessequence information describing acquired beneficial changes as afoundation for generating additional changes leading to subsequentchanges (improvements) in the desired properties of molecules encoded bythe character strings. Recursive GAGGS can be performed until strings ofcharacters are evolved to the point when the encoded polynucleotides andpolypeptides attain arbitrarily set levels of desired characteristics oruntil further changes in characteristics cannot be obtained (e.g.,enzyme turnover reached theoretical diffusion rate limit underconditions of a physical assay). Genetic algorithm parameters, genesynthesis methods and schemes, as well as physical assays and sequencedeconvolution methods can vary in each of the different rounds/cycles ofdirected evolution by recursive GAGGS.).

One particular advantage of this approach is that an initially random orpseudo random approach to library generation can become progressivelymore directed as information on activity levels becomes available. Forexample, any heuristic learning approach or neural network approachgradually becomes more efficient at selecting “correct” (active)sequences. A variety of such approaches are set forth below, includingprinciple component analysis, use of negative data, dataparameterization and the like.

Integration of GAGGS, DNA Shuffling and Other Directed EvolutionTechnologies.

GAGGS constitutes a self-sufficient and independent technology which canbe practiced regardless of DNA shuffling or any other available directedevolution methods. However, one or more rounds of GAGGS can be, andoften is, practiced in combination with physical shuffling of nucleicacids, and/or in combination with site directed mutagenesis, orerror-prone PCR (e.g. as alternating cycles of a directed evolutionprocess) or other diversity generation methods. GAGGS-generatedlibraries of polynucleotides can be subjected to nucleic acid shuffling,and polynucleotides found to have desired characteristics followingrounds of in silico and/or physical shuffling can be selected andsequenced to provide character strings to evaluate GAGGS processes or toform character strings for further GAGGS operations. Thus, GAGGS can beperformed as a stand-alone technology, or can be followed by shuffling,mutagenesis, random priming PCR, etc.

Where the methods of the invention entail performing physicalrecombination (“shuffling”) and screening or selection to evolveindividual genes, whole plasmids, viruses, multigene clusters, or evenwhole genomes, the techniques of the inventors and their co-workers areparticularly useful. For example, reiterative cycles of recombinationand screening/selection can be performed to further evolve the nucleicacids of interest which are generated by performing a GO on a characterstring (e.g., followed by synthesis of corresponding oligonucleotides,and gene generation/regeneration, e.g., by assembly PCR).

The following publications describe a variety of recursive recombinationprocedures and/or related diversity generation methods which can bepracticed in conjunction with the in silico processes of the invention:Stemmer, et al., (1999) “Molecular breeding of viruses for targeting andother clinical properties. Tumor Targeting” 4:1-4; Ness et al. (1999)“DNA Shuffling of subgenomic sequences of subtilisin” NatureBiotechnology 17:893-896; Chang et al. (1999) “Evolution of a cytokineusing DNA family shuffling” Nature Biotechnology 17:793-797; Minshulland Stemmer (1999) “Protein evolution by molecular breeding” CurrentOpinion in Chemical Biology 3:284-290; Christians et al. (1999)“Directed evolution of thymidine kinase for AZT phosphorylation usingDNA family shuffling” Nature Biotechnology 17:259-264; Crameri et al.(1998) “DNA shuffling of a family of genes from diverse speciesaccelerates directed evolution” Nature 391:288-291; Crameri et al.(1997) “Molecular evolution of an arsenate detoxification pathway by DNAshuffling,” Nature Biotechnology 15:436-438; Zhang et al. (1997)“Directed evolution of an effective fucosidase from a galactosidase byDNA shuffling and screening” Proceedings of the National Academy ofSciences, U.S.A. 94:4504-4509; Patten et al. (1997) “Applications of DNAShuffling to Pharmaceuticals and Vaccines” Current Opinion inBiotechnology 8:724-733; Crameri et al. (1996) “Construction andevolution of antibody-phage libraries by DNA shuffling” Nature Medicine2:100-103; Crameri et al. (1996) “Improved green fluorescent protein bymolecular evolution using DNA shuffling” Nature Biotechnology14:315-319; Gates et al. (1996) “Affinity selective isolation of ligandsfrom peptide libraries through display on a lac repressor ‘headpiecedimer’” Journal of Molecular Biology 255:373-386; Stemmer (1996) “SexualPCR and Assembly PCR” In: The Encyclopedia of Molecular Biology. VCHPublishers, New York. pp. 447-457; Crameri and Stemmer (1995)“Combinatorial multiple cassette mutagenesis creates all thepermutations of mutant and wildtype cassettes” BioTechniques 18:194-195;Stemmer et al., (1995) “Single-step assembly of a gene and entireplasmid form large numbers of oligodeoxyribonucleotides” Gene,164:49-53; Stemmer (1995) “The Evolution of Molecular Computation”Science 270: 1510; Stemmer (1995) “Searching Sequence Space”Bio/Technology 13:549-553; Stemmer (1994) “Rapid evolution of a proteinin vitro by DNA shuffling” Nature 370:389-391; and Stemmer (1994) “DNAshuffling by random fragmentation and reassembly: In vitro recombinationfor molecular evolution.” Proceedings of the National Academy ofSciences, U.S.A. 91:10747-1075:1.

Additional details regarding DNA shuffling methods are found in U.S.patents by the inventors and their co-workers, including: U.S. Pat. No.5,605,793 to Stemmer (Feb. 25, 1997), “METHODS FOR IN VITRORECOMBINATION;” U.S. Pat. No. 5,811,238 to Stemmer et al. (Sep. 92,1998) “METHODS FOR GENERATING POLYNUCLEOTIDES HAVING DESIREDCHARACTERISTICS BY ITERATIVE SELECTION AND RECOMBINATION;” U.S. Pat. No.5,830,721 to Stemmer et al. (Nov. 3, 1998), “DNA MUTAGENESIS BY RANDOMFRAGMENTATION AND REASSEMBLY;” U.S. Pat. No. 5,834,252 to Stemmer, etal. (Nov. 10, 1998) “END-COMPLEMENTARY POLYMERASE REACTION,” and U.S.Pat. No. 5,837,458 to Minshull, et al. (Nov. 17, 1998), “METHODS ANDCOMPOSITIONS FOR CELLULAR AND METABOLIC ENGINEERING.”

In addition, details and formats for nucleic acid shuffling are found ina variety of PCT and foreign patent application publications, including:Stemmer and Crameri, “DNA MUTAGENESIS BY RANDOM FRAGMENTATION ANDREASEMBLY” WO 95/22625; Stemmer and Lipschutz “END COMPLEMENTARYPOLYMERASE CHAIN REACTION” WO 96/33207; Stemmer and Crameri “METHODS FORGENERATING POLYNUCLEOTIDES HAVING DESIRED CHARACTERISTICS BY ITERATIVESELECTION AND RECOMBINATION” WO 97/0078; Minshul and Stemmer, “METHODSAND COMPOSITIONS FOR CELLULAR AND METABOLIC ENGINEERING” WO 97/35966;Punnonen et al. “TARGETING OF GENETIC VACCINE VECTORS” WO 99/41402;Punnonen et al. “ANTIGEN LIBRARY IMMUNIZATION” WO 99/41383; Punnonen etal. “GENETIC VACCINE VECTOR ENGINEERING” WO 99/41369; Punnonen et al.OPTIMIZATION OF IMMUNOMODULATORY PROPERTIES OF GENETIC VACCINES WO9941368; Stemmer and Crameri, “DNA MUTAGENESIS BY RANDOM FRAGMENTATIONAND REASSEMBILY” EP 0934999; Stemmer “EVOLVING CELLULAR DNA UPTAKE BYRECURSIVE SEQUENCE RECOMBINATION” EP 0932670; Stemmer et al.,“MODIFICATION OF VIRUS TROPISM AND HOST RANGE BY VIRAL GENOME SHUFFLING”WO 9923107; Apt et al., “HUMAN PAPILLOMAVIRUS VECTORS” WO 9921979; DelCardayre et al. “EVOLUTION OF WHOLE CELLS AND ORGANISMS BY RECURSIVESEQUENCE RECOMBINATION” WO 9831837; Patten and Stemmer, “METHODS ANDCOMPOSITIONS FOR POLYPEPTIDE ENGINEERING” WO 9827230; Stemmer et al. and“METHODS FOR OPTIMIZATION OF GENE THERAPY BY RECURSIVE SEQUENCESHUFFLING AND SELECTION” WO9813487.

Certain U.S. Applications provide additional details regarding DNAshuffling and related techniques, including “SHUFFLING OF CODON ALTEREDGENES” by Patten et al. filed Sep. 29, 1998, (U.S. Ser. No. 60/102,362),Jan. 29, 1999 (U.S. Ser. No. 60/117,729), and Sep. 28, 1999, U.S. Ser.No. PCT/US99/22588; “EVOLUTION OF WHOLE CELLS AND ORGANISMS BY RECURSIVESEQUENCE RECOMBINATION”, by del Cardyre et al. filed Jul. 15, 1999 (U.S.Ser. No. 09/354,922); “OLIGONUCLEOTIDE MEDIATED NUCLEIC ACIDRECOMBINATION” by Crameri et al., filed Feb. 5, 1999 (U.S. Ser. No.60/118,813) and filed Jun. 24, 1999 (U.S. Ser. No. 60/141,049) and filedSep. 28, 1999 (U.S. Ser. No. 09/408,392), and “USE OF CODON-BASEDOLIGONUCLEOTIDE SYNTHESIS FOR SYNTHETIC SHUFFLING” by Welch et al.,filed Sep. 28, 1999 (U.S. Ser. No. 09/408,393).

As review of the foregoing publications, patents, published applicationsand U.S. patent applications reveals, shuffling (or “recursiverecombination”) of nucleic acids to provide new nucleic acids withdesired properties can be carried out by a number of establishedmethods. Any of these methods are integrated with those of the presentinvention by incorporating nucleic acids corresponding to characterstrings produced by performing one or more GO on one or more selectedparental character string. Any of these methods can be adapted to thepresent invention to evolve GAGGS produced nucleic acids as discussedherein to produce new nucleic acids with improved properties. Both themethods of making such nucleic acids and the nucleic acids produced bythese methods are a feature of the invention.

In brief, at least 5 different general classes of recombination methodscan be performed (separately or in combination) in accordance with thepresent invention. First, nucleic acids such as those produced bysynthesis of sets of nucleic acids corresponding to character stringsproduced by GO manipulation of character strings, or availablehomologues of such sets, or both, can be recombined in vitro by any of avariety of techniques discussed in the references above, including e.g.,DNAse digestion of nucleic acids to be recombined followed by ligationand/or PCR reassembly of the nucleic acids. Second, sets of nucleicacids corresponding to character strings produced by GO manipulation ofcharacter strings, and/or available homologues of such sets, can berecursively recombined in vivo, e.g., by allowing recombination to occurbetween the nucleic acids while in cells. Third, whole cell genomerecombination methods can be used in which whole genomes of cells arerecombined, optionally including spiking of the genomic recombinationmixtures with desired library components such as with sets of nucleicacids corresponding to character strings produced by GO manipulation ofcharacter strings, or available homologues of such sets. Fourth,synthetic recombination methods can be used, in which oligonucleotidescorresponding to different homologues are synthesized and reassembled inPCR or ligation reactions which include oligonucleotides whichcorrespond to more than one parental nucleic acid, thereby generatingnew recombined nucleic acids. Oligonucleotides can be made by standardnucleotide addition methods, or can be made by tri-nucleotide syntheticapproaches. Filth, purely in silico methods of recombination can beeffected in which GOs are used in a computer to recombine sequencestrings which correspond to nucleic acid or proteins homologues. Theresulting recombined sequence strings are optionally converted intonucleic acids by synthesis of nucleic acids which correspond to therecombined sequences, e.g., in concert with oligonucleotidesynthesis/gene reassembly techniques. Any of the preceding generalrecombination formats, separately or together, can be practiced in areiterative fashion to generate a diverse set of recombinant nucleicacids.

The above references in conjunction with the present disclosure providethese and other basic recombination formats as well as manymodifications of these formats. Regardless of the format which is used,the nucleic acids of the invention can be recombined (with each other orwith related (or even unrelated) nucleic acids to produce a diverse setof recombinant nucleic acids, including homologous nucleic acids.

Other diversity generating approaches can also be used to modifycharacter strings or nucleic acids. Additional diversity can beintroduced into input or output nucleic acids by methods which result inthe alteration of individual nucleotides or groups of contiguous ornon-contiguous nucleotides, i.e., mutagenesis methods. Mutagenesismethods include, for example, recombination (PCT/US98/05223; Publ. No.WO98/42727); oligonucleotide-directed mutagenesis (for review see,Smith, Ann. Rev. Genet. 19: 423-462 (1985); Botstein and Shortle,Science 229: 1193-1201 (1985); Carter, Biochem. J. 237: 1-7 (1986);Kunkel, “The efficiency of oligonucleotide directed mutagenesis” inNucleic acids & Molecular Biology, Eckstein and Lilley, eds., SpringerVerlag, Berlin (1987)). Included among these methods areoligonucleotide-directed mutagenesis (Zoller and Smith, Nucl. Acids Res.10: 6487-6500 (1982), Methods in Enzymol. 100: 468-500 (1983), andMethods in Enzymol. 154: 329-350 (1987)) phosphothioate-modified DNAmutagenesis (Taylor et al., Nucl. Acids Res. 13: 8749-8764 (1985);Taylor et al., Nucl. Acids Res. 13: 8765-8787 (1985); Nakamaye andEckstein, Nucl. Acids Res. 14: 9679-9698 (1986); Sayers et al., Nucl.Acids Res. 16:791-802 (1988); Sayers et al., Nucl. Acids Res. 16:803-814 (1988)), mutagenesis using uracil-containing templates (Kunkel,Proc. Nat'l. Acad. Sci. USA 82: 488-492 (1985) and Kunkel et al.,Methods in Enzymol. 154:367-382)); mutagenesis using gapped duplex DNA(Kramer et al., Nucl. Acids Res. 12: 9441-9456 (1984); Kramer and Fritz,Methods in Enzymol. 154:350-367 (1987); Kramer et al., Nucl. Acids Res.16: 7207 (1988)); and Fritz et al., Nucl. Acids Res. 16: 6987-6999(1988)). Additional suitable methods include point mismatch repair(Kramer et al., Cell 38: 879-887 (1984)), mutagenesis usingrepair-deficient host strains (Carter et al., Nucl. Acids Res. 13:4431-4443 (1985); Carter, Methods in Enzymol. 154: 382-403 (1987)),deletion mutagenesis (Eghtedarzadeh and Henikoff, Nucl. Acids Res.14:5115 (1986)), restriction-selection and restriction-purification(Wells et al., Phil. Trans. R. Soc. Lond. A 317: 415-423 (1986)),mutagenesis by total gene synthesis (Nambiar et al., Science 223:1299-1301 (1984); Sakamar and Khorana, Nucl. Acids Res. 14: 6361-6372(1988); Wells et al., Gene 34:315-323 (1985); and Grundström et al.,Nucl. Acids Res. 13: 3305-3316 (1985). Kits for mutagenesis arecommercially available (e.g., Bio-Rad, Amersham International, AnglianBiotechnology).

Other diversity generation procedures are proposed in U.S. Pat. No.5,756,316; U.S. Pat. No. 5,965,408; Ostermeier et al. (1999) “Acombinatorial approach to hybrid enzymes independent of DNA homology”Nature Biotech 17:1205; U.S. Pat. No. 5,783,431; U.S. Pat. No.5,824,485; U.S. Pat. No. 5,958,672; Jirholt et al. (1998) “Exploitingsequence space: shuffling in vivo formed complementarity determiningregions into a master framework” Gene 215: 471; U.S. Pat. No. 5,939,250;WO 99/10539; WO 98/58085; WO 99/10539 and others. These diversitygenerating methods can be combined with each other or with shufflingreactions or in silico operations, in any combination selected by theuser, to produce nucleic acid diversity, which may be screened for usingany available screening method.

Following recombination or other diversification reactions, any nucleicacids which are produced can be selected for a desired activity. In thecontext of the present invention, this can include testing for andidentifying any detectable or assayable activity, by any relevant assayin the art. A variety of related (or even unrelated) properties can beassayed for, using any available assay.

Accordingly, a recombinant nucleic acid produced by recursivelyrecombining one or more polynucleotide of the invention (produced byGAGGS methods) with one or more additional nucleic acid forms a part ofthe invention. The one or more additional nucleic acid may includeanother polynucleotide of the invention; optionally, alternatively, orin addition, the one or more additional nucleic acid can include, e.g.,a nucleic acid encoding a naturally-occurring sequence or a subsequence,or any homologous sequence or subsequence.

The recombining steps can be performed in vivo, in vitro, or in silicoas described in more detail in the references above and herein. Alsoincluded in the invention is a cell containing any resulting recombinantnucleic acid, nucleic acid libraries produced by recursive recombinationof the nucleic acids set forth herein, and populations of cells,vectors, viruses, plasmids or the like comprising the library orcomprising any recombinant nucleic acid resulting from recombination (orrecursive recombination) of a nucleic acid as set forth herein withanother such nucleic acid, or an additional nucleic acid. Correspondingsequence strings in a database present in a computer system or computerreadable medium are a feature of the invention.

By way of example, a typical physical recombination procedure startswith at least two substrates that generally show at least some identityto each other (i.e., at least about 30%, 50%, 70%, 80% or 90% or moresequence identity), but differ from each other at certain positions(however, in purely in silico or cross-over oligonucleotide mediatedformats, nucleic acids can show little or no homology). For example, twoor more nucleic acids can be recombined herein. The differences betweenthe nucleic acids can be any type of mutation, for example,substitutions, insertions and deletions. Often, different segmentsdiffer from each other in about 1-20 positions. For recombination togenerate increased diversity relative to the starting materials, thestarting materials differ from each other in at least two nucleotidepositions. That is, if there are only two substrates, there should be atleast two divergent positions. If there are three substrates, forexample, one substrate can differ from the second at a single position,and the second can differ from the third at a different single position.Of course, even if only one initial character string is provided, any GOherein can be used to modify the nucleic acid to produce a diverse arrayof nucleic acids that can be screened for an activity of interest.

In physical shuffling procedures, starting DNA segments can be naturalvariants of each other, for example, allelic or species variants. Moretypically, they are derived from one or more homologous nucleic acidsequence. The segments can also be from nonallelic genes showing somedegree of structural and usually functional relatedness. The startingDNA segments can also be induced variants of each other. For example,one DNA segment can be produced by error-prone PCR replication of theother, or by substitution of a mutagenic cassette. Induced mutants canalso be prepared by propagating one (or both) of the segments in amutagenic strain. In these situations, strictly speaking, the second DNAsegment is not a single segment but a large family of related segments.The different segments forming the starting materials are often the samelength or substantially the same length. However, this need not be thecase; for example, one segment can be a subsequence of another. Thesegments can be present as part of larger molecules, such as vectors, orcan be in isolated form. In one option, the nucleic acids of interestare derived from DE by GAGGS.

Codon-Varied Oligonucleotide Methods

Codon-varied oligonucleotides are oligonucleotides, similar in sequencebut with one or more base variations, where the variations correspond toat least one encoded amino acid difference. They can be synthesizedutilizing tri-nucleotide, i.e., codon-based phosphoramidite couplingchemistry, in which tri-nucleotide phosphoramidites representing codonsfor all 20 amino acids are used to introduce entire codons intooligonucleotide sequences synthesized by this solid-phase technique.Preferably, all of the oligonucleotides of a selected length (e.g.,about 20, 30, 40, 50, 60, 70, 80, 90, or 100 or more nucleotides) whichincorporate the chosen nucleic acid sequences are synthesized. In thepresent invention, codon-varied oligonucleotide sequences can be basedupon sequences from a selected set of nucleic acids, generated by any ofthe approaches noted herein. Further details regarding tri-nucleotidesynthesis are found in U.S. Ser. No. 09/408,393 “USE OF CODON VARIEDOLIGONUCLEOTIDE SYNTHESIS FOR SYNTHETIC SHUFFLING” by Welch, et al.,filed Sep. 28, 1999.

Oligonucleotides can be made by standard nucleotide addition methods, orcan be made by tri-nucleotide synthetic approaches. An advantage ofselecting changes which correspond to encoded amino acid differences isthat the modification of triplets of codons results in fewer frameshifts(and, therefore, likely fewer inactive library members). Also, synthesiswhich focuses on codon modification, rather than simply on basevariation, reduces the total number of oligos which are needed for asynthesis protocol.

Oligo Sets

In general, sets of oligos can be combined for assembly in manydifferent formats and different combinations schemes to effectcorrelation with genetic events and operators at the physical level.

As noted, overlapping sets of oligonucleotides can be synthesized andthen hybridized and elongated to form full-length nucleic acids. A fulllength nucleic acid is any nucleic acid desired by an investigator whichis longer than the oligos which are used in the gene reconstructionmethods. This can correspond to any percentage of a naturally occurringfull length sequence, e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or90% or more of the corresponding natural sequence.

Oligo sets often have at least about 5, sometimes about 10, often about15, generally about 20, or more, nucleotide overlap sequences tofacilitate gene reconstruction. Oligo sets are optionally simplified forgene reconstruction purposes where regions of fortuitous overlap arepresent, i.e., where repetitive sequence elements are present ordesigned into a gene sequence to be synthesized. Lengths of oligos in aset can be the same or different, as can the regions of sequenceoverlap. To facilitate hybridization and elongation (e.g., during cyclesof PCR), overlap regions are optionally designed with similar meltingtemperatures.

Parental sequences can be gridded (conceptually or physically) and thecommon sequences used to select common sequence oligos, therebycombining oligo members into one or more sets to reduce the number ofoligos required for making full-length nucleic acids. Similarly,oligonucleotides with some sequence similarity can be generated bypooled and/or split synthesis where pools of oligos under synthesis aresplit into different pools during the addition of heterologous bases,optionally followed by rejoined synthesis steps (pooling) at subsequentstages where the same additions to the oligos are required. In Oligoshuffling formats, heterologous oligos corresponding to many differentparents can be split and rejoined during synthesis. In simple degeneratesynthetic approaches, more than one nucleobase can be added duringsingle synthetic steps to produce two or more variations in sequence intwo or more resulting oligonucleotides. The relative percentage ofnucleobase addition can be controlled to bias synthesis towards one ormore parental sequence. Similarly, partial generacy can be practiced toprevent the insertion of stop codons during degenerated oligonucleotidesynthesis.

Oligos which correspond to similar subsequences from different parentscan be the same length or different, depending on the subsequences.Thus, in split and pooled formats, some oligos are optionally notelongated during every synthetic step (to avoid frame-shifting, someoligos are not elongated for the steps corresponding to one or morecodon).

When constructing oligos, crossover oligos can be constructed at one ormore point of difference between two or more parental sequences (a basechange or other difference is a genetic locus which can be treated as apoint for a crossover event). The crossover oligos have a region ofsequence identity to a first parental sequence, followed by a region ofidentity to a second parental sequence, with crossover point occurringat the locus. For example, every natural mutation can be a cross overpoint.

Another way of biasing sequence recombination is to spike a mixture ofoligonucleotides with fragments of one or more parental nucleic acid (ifmore than one parental nucleic acid is fragmented, the resultingsegments can be spiked into a recombination mixture at differentfrequencies to bias recombination outcomes towards one or more parent).Recombination events can also be engineered simply by omitting one ormore oligonucleotide corresponding to one or more parent from arecombination mixture.

In addition to the use of families of related oligonucleotides,diversity can be modulated by the addition of selected, pseudo-random orrandom oligos to elongation mixture, which can be used to bias theresulting full-length sequences. Similarly, mutagenic or non-mutagenicconditions can be selected for PCR elongation, resulting in more or lessdiverse libraries of full length nucleic acids.

In addition to mixing oligo sets which correspond to different parentsin the elongation mixture, oligo sets which correspond to just oneparent can be elongated to reconstruct that parent. In either case, anyresulting full-length sequence can be fragmented and recombined, as inthe DNA shuffling methods noted in the references cited herein.

Many other oligonucleotide sets and synthetic variations which can becorrelated to genetic events and operators at the physical level arefound in “OLIGONUCLEOTIDE MEDIATED NUCLEIC ACID RECOMBINATION” byCrameri et al., filed Feb. 5, 1999 (U.S. Ser. No. 60/118,813) and filedJun. 24, 1999 (U.S. Ser. No. 60/141,049) and filed Sep. 28, 1999 (U.S.Ser. No. 09/408,392) and “USE OF CODON-BASED OLIGONUCLEOTIDE SYNTHESISFOR SYNTHETIC SHUFFLING” by Welch et al., filed Sep. 28, 1999 (U.S. Ser.No. 09/408,393).

Targets for Codon Modification and Shuffling

Essentially any nucleic acid can be shuffled using the GAGGS methodsherein. No attempt is made herein to identify the hundreds of thousandsof known nucleic acids. Common sequence repositories for known proteinsinclude GenBank EMBL, DDBJ and the NCBI. Other repositories can easilybe identified by searching the internet.

One class of preferred targets for GAGGS methods includes nucleic acidsencoding therapeutic proteins such as erythropoietin (EPO), insulin,peptide hormones such as human growth hormone; growth factors andcytokines such as epithelial Neutrophil Activating Peptide-78,GRO∀/MGSA, GRO∃, GRO(, MIP-1∀, MIP-1&, MCP-1, epidermal growth factor,fibroblast growth factor, hepatocyte growth factor, insulin-like growthfactor, the interferons, the interleukins, keratinocyte growth factor,leukemia inhibitory factor, oncostatin M, PD-ECSF, PDGF, pleiotropin,SCF, c-kit ligand, VEGEF, G-CSF etc. Many of these proteins and theircorresponding coding nucleic acids are commercially available (See e.g.,the Sigma BioSciences 1997 catalogue and price list), and, in any case,the corresponding genes are well-known.

Another class of preferred targets for GAGGS are transcription andexpression activators. Example transcriptional and expression activatorsinclude genes and proteins that modulate cell growth, differentiation,regulation, or the like. Expression and transcriptional activators arefound in prokaryotes, viruses, and eukaryotes, including fungi, plants,and animals, including mammals, providing a wide range of therapeutictargets. It will be appreciated that expression and transcriptionalactivators regulate transcription by many mechanisms, e.g., by bindingto receptors, stimulating a signal transduction cascade, regulatingexpression of transcription factors, binding to promoters and enhancers,binding to proteins that bind to promoters and enhancers, unwinding DNA,splicing pre-mRNA, polyadenylating RNA, and degrading RNA. Expressionactivators include cytokines, inflammatory molecules, growth factors,their receptors, and oncogene products, e.g., interleukins (e.g., IL-1,IL-2, IL-8, etc.), interferons, FGF, IGF-I, IGF-II, FGF, PDGF, TNF,TGF-∀, TGF-∃, EGF, KGF, SCF/c-Kit, CD40L/CD40, VLA-4/VCAM-1,ICAM-1/LFA-1, and hyalurin/CD44; signal transduction molecules andcorresponding oncogene products, e.g., Mos, Ras, Raf, and Met; andtranscriptional activators and suppressors, e.g., p53, Tat, Fos, Myc,Jun, Myb, Rel, and steroid hormone receptors such as those for estrogen,progesterone, testosterone, aldosterone, the LDL receptor ligand andcorticosterone.

Similarly, proteins from infectious organisms for possible vaccineapplications, described in more detail below, including infectiousfungi, e.g., Aspergillus, Candida species; bacteria, particularly E.coli, which serves a model for pathogenic bacteria, as well as medicallyimportant bacteria such as Staphylococci (e.g., aureus), Streptococci(e.g., pneumoniae), Clostridia (e.g., perfingens), Neisseria (e.g.,gonorrhoea), Enterobacteriaceae (e.g., coli), Helicobacter (e.g.,pylori), Vibrio (e.g., cholerae), Capylobacter (e.g., jejuni),Pseudomonas (e.g., aeruginosa), Hemeophilus (e.g., inifluenzae),Bordetella (e.g., pertussis), Mycoplasma (e.g., pneumoniae), Ureaplasma(e.g., urealyticum), Legionella (e.g., pneumophila), Spirochetes (e.g.,Treponema, Leptospira, and Borrelia), Mycobacteria (e.g., tuberculosis,smegmatis), Actinomyces (e.g., israelii), Nocardia (e.g., asteroides),Chlamydia (e.g., trachomatis), Rickettsia, Coxiella, Ehrilichia,Rochaliniaea, Bracella, Yersinia, Fracisella, and Pasteurella; protozoasuch as sporozoa (e.g., Plasmodia), rhizopods (e.g., Entamoeba) andflagellates (Trypanosoma, Leishmania, Trichomonas, Giardia, etc.);viruses such as (+) RNA viruses (examples include Poxviruses e.g.,vaccinia; Picornaviruses, e.g. polio; Togaviruses, e.g., rubella;Flaviviruses, e.g., HCV; and Coronaviruses), (−) RNA viruses (examplesinclude Rhabdoviruses, e.g., VSV; Paramyxovimses, e.g., RSV;Orthomyxovimses, e.g., influenza; Bunyaviruses; and Arenaviruses), dsDNAviruses (Reoviruses, for example), RNA to DNA viruses, i.e.,Retroviruses, e.g., especially HIV and HTLV, and certain DNA to RNAviruses such as Hepatitis B virus.

Other nucleic acids encoding proteins relevant to non-medical uses, suchas inhibitors of transcription or toxins of crop pests e.g., insects,fungi, weed plants, and the like, are also preferred targets for GAGGS.Industrially important enzymes such as monooxygenases, proteases,nucleases, and lipases are also preferred targets. As an example,subtilisin can be evolved by shuffling selected forms of the gene forsubtilisin (von der Osten et al., J. Biotechnol. 28:55-68 (1993) providea subtilisin coding nucleic acid). Proteins which aid in folding such asthe chaperonins are also preferred.

Preferred known genes suitable for codon alteration and shuffling alsoinclude the following: Alpha-1 antitrypsin, Angiostatin, Antihemolyticfactor, Apolipoprotein, Apoprotein, Atrial natriuretic factor, Atrialnatriuretic polypeptide, Atrial peptides, C-X-C chemokines (e.g.,T39765, NAP-2, ENA-78, Gro-a, Gro-b, Gro-c, IP-10, GCP-2, NAP-4, SDF-1,PF4, MIG), Calcitonin, CC chemokines (e.g., Monocyte chemoattractantprotein-1, Monocyte chemoattractant protein-2, Monocyte chemoattractantprotein-3, Monocyte inflammatory protein-1 alpha, Monocyte inflammatoryprotein-1 beta, RANTES, 1309, R83915, R91733, HCC1, T58847, D31065,T64262), CD40 ligand, Collagen, Colony stimulating factor (CSF),Complement factor 5a, Complement inhibitor, Complement receptor 1,Factor IX, Factor VII, Factor VIII, Factor X, Fibrinogen, Fibronectin,Glucocerebrosidase, Gonadotropin, Hedgehog proteins (e.g., Sonic,Indian, Desert), Hemoglobin (for blood substitute; forradiosensitization), Hirudin, Human serum albumin, Lactoferrin,Luciferase, Neurturin, Neutrophil inhibitory factor (NIF), Osteogenicprotein, Parathyroid hormone, Protein A, Protein G, Relaxin, Renin,Salmon calcitonin, Salmon growth hormone, Soluble complement receptor I,Soluble I-CAM 1, Soluble interleukin receptors (IL-1, 2, 3, 4, 5, 6, 7,9, 10, 11, 12, 13, 14, 15), Soluble TNF receptor, Somatomedin,Somatostatin, Somatotropin, Streptokinase, Superantigens, i.e.,Staphylococcal enterotoxins (SEA, SEB, SEC1, SEC2, SEC3, SED, SEE),Toxic shock syndrome toxin (TSST-1), Exfoliating toxins A and B,Pyrogenic exotoxins A, B, and C, and M. arthritides mitogen, Superoxidedismutase, Thymosin alpha 1, Tissue plasminogen activator, Tumornecrosis factor beta (TNF beta), Tumor necrosis factor receptor (TNFR),Tumor necrosis factor-alpha (TNF alpha) and Urokinase.

Other preferred genes for shuffling include p450s (these enzymesrepresent a very diverse set of natural diversity and catalyze manyimportant reactions); see, e.g., Ortiz de Montellano (ed.) (1995)Cytochrome P450 Structure Mechanism and Biochemistry, Second EditionPlenum Press (New York and London) and the references cited therein foran introduction to cytochrome P450. Other monooxygenases, as well asdioxygenases, acyl transferases (cis-diol), halogenated hydrocarbondehalogenases, methyl transferases, terpene synthetases, and the like,can be shuffled.

The Uses of Consensus Genes in Directed Evolution, Including “Diplomacy”

One of the factors involved in standard family shuffling of parentalgenes is the extent of identity of the genes to be physicallyrecombined. Genes of limited identity are difficult to recombine withoutcross-over oligonucleotides, and often result in shuffled librarieshaving unacceptable knockout rates, or no chimera formation, noactivity, no functional library, etc. In one aspect, the presentinvention overcomes this difficulty by providing for in silico design ofa “diplomat” sequence which has an intermediate level of homology toeach of the sequences to be recombined, thereby facilitating cross-overevents between the sequences and facilitating chimera formation. Thisdiplomat sequence can be a character string produced by any of a varietyof GO to establish intermediate sequence similarity in the diplomatsequence as compared to the sequences to be recombined, including byalignment of the sequences to select a consensus sequence, codonmodification to optimize similarity between diverse nucleic acids, orthe like.

As noted, one way in which to design a diplomat sequence is simply toselect a consensus sequence, e.g., using any of the approaches herein.The consensus sequence is generated by comparison and lining-up/pile-upof a family of genes (DNA consensus), or of amino acid sequenceline-up/pile-up (aa consensus). In the latter case, the amino acidconsensus sequence are optionally back-translated using a desirablecodon bias to further enhance homology, or to enhance host organism forexpression, or to select for alternate codon usages in order to enableaccess to alternative sets of amino acid codons. One can also usedifferent subsets of gene families to generate consensus sequences.

Furthermore, the consensus sequence itself may encode an improvedenzyme. This has been observed elsewhere (e.g. presentation atInternational Conference “Enzyme Opportunities on the Next Millenium”,Chicago, Ill., May 5-7, by Dr. Luis Pasamontes, Roche Vitamins, Inc., on“Development of Heat Stable Phytase”—a consensus phytase had an increaseof 16 degrees C. in thermostability). Another example of a consensusprotein having improved properties is consensus Interferon (IFN-con1).

An additional approach is to design consensus sequences to control(minimize, maximize, direct, etc.) construction of cross-overoligonucleotides, e.g., as discussed supra.

Accordingly, separately or in conjunction with any of the techniquesherein, diplomat sequences can be designed using selected GO criteriaand, optionally, physically synthesized and shuffled using any of thetechniques herein.

Example Processing Steps for Reverse Translation and Oligo Design

Automatic processing steps (e.g., performed in a digital system asdescribed herein) that perform the following functions facilitateselection of oligonucleotides in synthetic shuffling techniques herein.

For example, the system can include an instruction set which permitsinputting of amino acid sequences of a family of proteins of interest ofinterest.

These sequences are back-translated with any desired codon usageparameters, e.g., optimal usage parameters for one or more organism tobe used for expression, or to optimize sequence alignments to facilitaterecombination, or both. For example, codon usage can be selected formultiple expression hosts, e.g. E. coli and S. cerevisiae. In somecases, simply optimizing codon usage for expression in a host cell willresult in making homologous sequences more similar, as they will losetheir natural species codon bias.

Sequences are aligned, and a consensus sequence is produced, optionallyshowing degenerate codons.

Oligonucleotides are designed for synthetic construction of one or morecorresponding synthetic nucleic acid for shuffling. Input parameters onoligonucleotide design include minimum and maximum lengths, minimumlength of identical sequence at the ends, maximum degeneracy peroligonucleotide, length of oligo overlap, etc.

As noted, an alternative to back-translation to achieve optimal codonusage for expression in a particular organism is to back-translatesequences to optimize nucleotide homology between family members. Forexample, amino acid sequences are aligned. All possible codons for eachamino acid are determined and codons that minimize differences betweenthe family of aligned sequences are chosen at each position.

Use of Family Shuffling to Identify Structural Motifs Confering SpecificProtein Properties

It is often of interest to identify regions of a protein that areresponsible for specific properties, to facilitate functionalmanipulation and design of related proteins. This identification istraditionally done using structural information, usually obtained bybiophysical techniques such as X-ray crystallography. The presentinvention provides an alternative method in which variants are obtainedand analyzed for specific properties which are then correlated withsequence motifs.

The sequences of naturally occurring enzymes that catalyze similar oreven identical reactions can vary widely: sequences may be only 50%identical or less. While a family of such enzymes can each catalyze anessentially identical reaction, other properties of these enzymes maydiffer significantly. These include physical properties such asstability to temperature and organic solvents, pH optima, solubility,the ability to retain activity when immobilized, ease of expression indifferent host systems, etc. They also include catalytic propertiesincluding activity (k_(cat) and K_(m)), the range of substrates acceptedand even the chemistries performed.

The method described here can also be applied to non-catalytic proteins(i.e. ligands such as cytokines) and even nucleic acid sequences (suchas promoters that may be inducible by a number of different ligands),wherever multiple functional dimensions are encoded by a family ofhomologous sequences.

Because of the divergence between enzymes with similar catalyticfunctions, it is not usually possible to correlate specific propertieswith individual amino acids at certain positions, as there are simplytoo many amino acid differences. However, libraries of variants can beprepared from a family of homologous natural sequences by DNA familyshuffling. These libraries contain the diversity of the original set ofsequences, in a large number of different combinations. If individualsfrom the library are then tested under a specific set of conditions fora particular property, the optimal combinations of sequences from theparental set for those conditions can be determined.

If the assay conditions are then altered in only one parameter,different individuals from the library will be identified as the bestperformers. Because the screening conditions are very similar, mostamino acids are conserved between the two sets of best performers.Comparisons of the sequences (e.g., in silico) of the best enzymes underthe two different conditions identifies the sequence differencesresponsible for the differences in performance. Principal componentanalysis is a powerful tool to use for identifying sequences conferringa particular property. For example, Partek Incorporated (St. Peters,Mo.; partek.com, on the world wide web) provides software for patternrecognition (e.g., which provide Partek Pro 2000 Pattern RecognitionSoftware) which can be applied to genetic algorithms for multivariatedata analysis, interactive visualization, variable selection, neural &statistical modeling. Relationships can be analyzed, e.g., by PrincipalComponents Analysis (PCA) mapped scatterplots and biplots,Multi-Dimensional Scaling (MDS), mapped scatterplots, Star plots, etc.

Once sequence motifs have been identified, proteins are manipulated in,e.g., any of a number of ways. For example, identified changes areoptionally deliberately introduced into other sequence backrounds.Sequences conferring different specific properties can be combined.Identified sequence regions of importance for a specific function can betargeted for more thorough investigation, for example by completerandomization using degenerate oligonucleotides, e.g., selected by insilico processes.

Identification of Parental Contributors to Chimeras Produced by FamilyShuffling

This example provides a method for the identification of parentalcontributors to chimeras produced by family shuffling.

The method takes as an input the sequences of parental genes, and thesequences of chimeras, and compares each chimera with each parent. Itthen builds sequence and graphical maps of each chimera, indicating theparental source of each chimeric fragment. Correlation of this withfunctional data permits identification of parents that contribute tospecific properties and thereby facilitates the selection of parents fornew more focused libraries which can be made by any of the methods notedherein, and screened for any desired functional property.

In one simple example, family 3 and 4 genes contribute to an activityat, e.g., pH 5.5, while family 1 and 2 genes are better at pH 10. Thus,for an application at low pH, the parental composition to create alibrary would be biased towards 3 and 4, while for high pH apredominantly 1 and 2-based library would be appropriate. Thus, a GO canbe implemented which selects oligonucleotides for gene reconstructionpredominantly from families 3 and 4. Additional details regarding geneblending methods utilized in oligonucleotide shuffling are found in“OLIGONUCLEOTIDE MEDIATED NUCLEIC ACID RECOMBINATION” by Crameri et al.,filed Feb. 5, 1999 (U.S. Ser. No. 60/118,813) and filed Jun. 24, 1999(U.S. Ser. No. 60/141,049) and filed Sep. 28, 1999 (U.S. Ser. No.09/408,392).

Gene blending is similar to principal component analysis (PCA) foridentification of specific sequence motifs. Principal component analysis(PCA) is a mathematical procedure that transforms a number of (possibly)correlated variables into a (smaller) number of uncorrelated variablescalled “principal components.” The first principal component accountsfor as much of the variability in the data as possible, and eachsucceeding component accounts for as much of the remaining variabilityas possible. Traditionally, principal component analysis is performed ona square symmetric SSCP (pure sums of squares and cross products)matrix, covariance (scaled sums of squares and cross products) matrix,or correlation (sums of squares and cross products from standardizeddata) matrix. The analysis results for objects of type SSCP andcovariance are similar. A correlation object is used when the variancesof individual variates differ substantially or the units of measurementof the individual variates differ. Objectives of principal componentanalysis include, e.g., to discover or to reduce the dimensionality of adata set, to identify new meaningful underlying variables, and the like.

The main difference is that the present operation gives informationabout which parents to use in a mix (and so directs construction of newnatural gene-based libraries), while PCA identifies specific motifs andso is well suited for more general synthetic shuffling, with identifieddiscrete regions being altered in either a directed or randomized way.

Partek (PCA) software discussed above has an “experimental design”component, that identifies variables that appear to have an effect on aspecific function. As applied to the present example, this is useful inan iterative process in which a family library is constructed andscreened and the resulting chimeras analyzed for functional correlationswith sequence variations. This is used to predict sequence regions for aparticular function, and a library is selected in silico by any desiredGO directed changes of the region which correlates to functionalactivity. A focused library which has diversity in those regions isconstructed, e.g., by oligonucleotide synthetic methods as describedherein. The resulting library members (chimeras) are analyzed forfunctional correlations with sequence variations. This approach focusesthe search of variation in sequence space on the most relevant regionsof a protein or other relevant molecule.

After sequences which are active are deconvoluted, the resultingsequence information is used to refine further predictions for in silicooperations, e.g., in a neural net training approach.

For example, neural net approaches can be coupled to geneticalgorithm-type programming. For example, NNUGA (Neural Network UsingGenetic Algorithms) is an available program (cs.bgu.ac.i1/˜omri/NNUGA/,on the world wide web) which couples neural networks and geneticalgorithms. An introduction to neural networks can be found, e.g., inKevin Gurney (1999) An Introduction to Neural Networks, UCL Press, 1Gunpowder Square, London EC4A 3DE, UK. and atshef.ac.uk/psychology/gurney/notes/index.html (on the world wide web)additional useful neural network references include those noted above inregard to genetic algorithms and, e.g., Christopher M. Bishop (1995)Neural Networks for Pattern Recognition Oxford Univ Press; ISBN:0198538642; Brian D. Ripley, N. L. Hjort (Contributor) (1995) PatternRecognition and Neural Networks Cambridge Univ Pr (Short); ISBN:0521460867.

In Silico Shuffling Incorporating Rational and/or Statistical Methods

One aspect of the present invention is the coupling of logical filteringmechanisms to nucleic acid or polypeptide sequences in silico and, e.g.,random physical shuffling of logically “filtered” nucleic acids orpolypeptides. As discussed throughout, in silico approaches can be usedto apply any desired criteria to selection of recombination events,which are optionally coupled to physical shuffling processes to generateselected, random or pseudo-random recombined physical sequences forsubsequent activity selection. Three basic logical GO filters arediscussed herein in detail, though others will be apparent to one ofskill.

First, structural considerations can be used to design logical filterswhich preserve or eliminate any structural feature of interest in anucleic acid or encoded protein (as discussed herein, direct design andsynthesis of recombinant proteins without nucleic acid intermediates canbe performed, but, for simplicity of illustration, the followingdiscussion generally describes the use of nucleic acids to generateproteins). Such structural criteria include e.g., energy minimizationcalculations, combinatorial automated protein design algorithms,sequence motifs, application of GAs or GOs to structural and/or sequencecriteria, and, e.g., structural information based upon any availablestructural or modeling data. For example, structural data is provided,e.g., as derived from physical protein (or nucleic acid) analysis (e.g.,crystal structure, nmr, epr, circular dichrosim, intrinsic fluorescence,mass spectrometry, and any of the myriad other available structuralanalysis methods) or by any structural modeling method. It will also beappreciated that modeling and physical structure information areoptionally used in conjunction and that cycles of physical informationanalysis, modeling and application of this information in one or more GOin silico can be performed in conjunction. Indeed, certain combinatorialprotein design algorithms themselves rely on cycles of experimentationand design (such approaches are discussed in more detail below).

Structural considerations can include, e.g., a logical filter (e.g., aspart of any GO) which maintains or disrupts any interaction of two ormore amino acids in a protein sequence. Where the filter is set tomaintain structural features, the two or more amino acids will beselected to maintain their physical relationship and will, therefore,show co-variance in subsequent recombinants. That is, cross-overs orother GOs are selected by the filter such that the physical-structuralrelationship of the two or more amino acids is linked or maintained (ordisrupted, if desired). Generally, physical linkages can include anysort of structural element maintenance or disruption, includingmaintaining or disrupting physical distance relationships, energyminimization relationships, etc. It is, of course, useful to maintain ordisrupt particular structures to derive similar or fundamentallydifferent nucleic acids or proteins from a set of parental nucleic acidsor proteins.

Another aspect that is related to function is the constraint to fold.Several experimental studies have focused on understanding intermediatesin the folding pathway (e.g., Bai et. al, Science 1995 269:192-197).Thus, not only the structural considerations of the final nativestructure can be used to provide logical filters, but also intermediatesduring the folding pathway can be used for this purpose. Thisinformation can be used to make sure that any residue that may beimportant for the folding pathway can be retained.

Second, statistical considerations can be applied to provide any of avariety of filters for identifying relationships between nucleic acid orprotein sequences. Many approaches are set forth herein, e.g., whichdetermine which nucleic acids or amino acids are useful for activitycriteria. e.g., based upon consideration of evolutionary algorithms thatexamine evolutionary relationships; for example, sequence conservation,homoplasy (independent convergent evolution that retains similaritybetween sequences), as well as learning algorithms that examine theeffects of sequence alterations over one or more design cycles, Markovmodeling, principle component analysis and the like can be used. Forexample, independent convergent evolution that retains similaritybetween sequences is called homoplasy. Statistical methods can identifyhomoplasic sites in a protein which can extend understanding of theprotein structure by separating residues that are evolving to retainparent property. (See, e.g., Meyer. A Novartis Found Symp 1999,222:141-153).

Third, combinations of structural and statistical approaches can beused. These can either constitute separate filters, or can be applied ina single complex logical filter. That is, it will be appreciated thatmany statistical observations are, ultimately, derived from structuralrelationships. Thus, algorithms that take account of both structuralinformation and empirical statistical observations can be used aslogical filters. Here again, complex cycles of protein structuralanalysis, modeling, GO application to design sequences in silico,recombinant nucleic acid/protein generation, screening and statisticalanalysis can be performed recursively, with each step in the processproviding information that is useful in subsequent steps. For example,statistical analysis of empirical results can suggest that certainstructural events or features are relevant, and can be used to refinemodeling prediction and to elucidate structural analysis, and thisinformation can be applied to the next cycle of GO application in silicoto select additional cross-over points or to apply any other GO ofinterest.

Several of the preceding and following sections give details on thesevarious filtering approaches for the coupling of rational orsemi-rational design criteria to random or semi-random recombinationmethods.

Coupling of Rational Protein Design and Shuffling

As noted, one aspect of the present invention is the use of any form ofstructural information in applying a logical filter to design cross-overpoints, or, for that matter, to design or modify any other GO as notedherein. These GOs include, without limitation, mutation of one or moreparental character strings or one or more character string subsequences,multiplication of one or more parental character strings or one or morecharacter string subsequences, fragmentation of one or more parentalcharacter strings or one or more character string subsequences,crossover between any of the one or more parental character strings orone or more character string subsequences or an additional characterstring, ligation of the one or more parental character strings or one ormore character string subsequences, an elitism calculation, acalculation of sequence homology or sequence similarity of alignedstrings, a recursive use of one or more genetic operator for evolutionof character strings, application of a randomness operator to the one ormore parental character strings or the one or more character stringsubsequences, a deletion mutation of the one or more parental characterstrings or one or more character string subsequences, an insertionmutation into the one or more parental character strings or one or moreof character string subsequences, subtraction of the of the one or moreparental character strings or one or more character string subsequenceswith an inactive sequence, selection of the of the one or more parentalcharacter strings or one or more character string subsequences with anactive sequence, and death of the one or more parental character stringsor one or more of character string subsequences. That is, any GO caninclude a component which maintains, removes, modifies, or in any waymodulates one or more structural relationship during application of theGO in a GA.

Protein design cycles, involving cycling between theory and experiment,has led to recent advances in rational protein design (reviewed, e.g.,in Street and Mayo (1999) “Computational Protein Design” Structure withFolding and Design 7(5):R105—R109). As noted above, performing iterativeor recursive cycles of design, recombinant generation and screening andstatistical analysis can be applied to the present invention (of course,these approaches can be used individually and in a variety ofcombinations); moreover, the combination of protein design cycles withrandom or partly random recombination processes can be used not only toproduce proteins and nucleic acids of interest, but also in a learningapproach to improve the protein design tools themselves.

With respect to modeling and structural analysis, a reductionistapproach, in which protein positions are classified by their localenvironments, has aided development of appropriate energy expressions(reviewed in Street and Mayo (1999) “Computational Protein Design”Structure with Folding and Design 7(5):R105-R109). For examples ofcycles of modeling and structural analysis, See, e.g., mayo.caltech.edu/(on the world wide web); Gordon and Mayo (1999) “Brancht-and-Terminate:A Combinatorial Optimization Algorithm for Protein Design” Structurewith Folding and Design 7(9): 1089-1098; Street and Mayo (1999)“Intrinsic β-sheet Propensities Result from van der Waals InteractionsBetween Side Chains and the Local Backbone” Proc. Natl. Acad. Sci. USA,96, 9074-9076; Gordon et al. (1999) “Energy Functions for ProteinDesign” Current Opinion in Structural Biology 9(4):509-513; Street andMayo (1999) “Computational Protein Design” Structure with Folding andDesign 7(5):R105-R109; Strop and Mayo (1999) “Rubredoxin Variant FoldsWithout Iron” J. Am. Chem. Soc. 121(11):2341-2345; Gordon and Mayo(1998) “Radical Performance Enhancements for Combinatorial OptimizationAlgorithms based on the Dead-End Elimination Theorem” J. Comp. Chem.19:1505-1514; Malakauskas and Mayo (1998) “Design, Structure, andStability of a Hyperthermophilic Protein Variant” Nature Struct. Biol.5:470; Street and Mayo (1998) “Pairwise Calculation of ProteinSolvent-Accessible Surface Areas” Folding & Design 3: 253-258. Dahiyatand Mayo (1997) “De Novo Protein Design: Fully Automated SequenceSelection” Science 278:82-87; Dahiyat and Mayo (1997) “Probing the Roleof Packing Specificity in Protein Design” Proc. Natl. Acad. Sci. USA94:10172-10177; Haney et al. (1997) “Structural basis forthermostability and identification of potential active site residues foradenylate kinases from the archaeal genus Methanococcus” Proteins 28(1):117-30; and, Dahiyat et al. (1997) “Automated Design of the SurfacePositions of Protein Helices” Prot. Sci. 6:1333-1337; Dahiyat et al.(1997) “De Novo Protein Design: Towards Fully Automated SequenceSelection”J. Mol. Biol. 273:789-796. Protein design programs can be usedto build or modify proteins with any selected set of design criteria andthese design criteria can be used as filters for any GO as noted herein,and/or in recursive cycles of design (by modeling or structuralanalysis), in silico GO or GA application, and/or physical recombinationof nucleic acids of interest.

For example, in Street and Mayo (1999) “Intrinsic β-sheet PropensitiesResult from van der Waals Interactions Between Side Chains and the LocalBackbone” Proc. Natl. Acad. Sci. USA, 96, 9074-9076, the intrinsicsecondary structure-forming propensities of naturally occurring aminoacids were measured experimentally in host-guest studies andstatistically by examination of a protein structure databank. Asdescribed, the linkage between dipeptides was modeled using a van derWaals energy function and derived Ramachandran plots for each of theamino acids. These data were used to determine the entropy and Helmholtzfree-energy region of φ-Ψ space. A cause of intrinsic ∃-sheet propensitywas found to be the avoidance of steric clashes between an amino acidside chain and its local backbone. Standard implementations of coulombicand salvation effects were determined to be of less significance. Thus,in addition to assigning linkages between amino acids or nucleotides insecondary and tertiary structures of proteins, linkages can be assignede.g., at the dipeptide level (or any other level) to providestructure/design criteria. Again, these linkages can be selected for ordisrupted, depending on the desired outcome, in selection of cross-overevents or other GOs, e.g., in silico, according to the presentinvention. After application of the relevant GO, physicalsynthesis/recombination of relevant nucleic acids is optionallyperformed, as discussed herein. This process can be repeated recursivelyto improve both the nucleic acids/proteins of interest and to improvethe relevant design or GO tools, e.g., by applying a learning heuristicto the analysis of results.

In a similar fashion, the design of surface positions of proteins (e.g.,external helicies) has been considered (Dahiyat et al. (1997) “AutomatedDesign of the Surface Positions of Protein Helices” Prot. Sci.6:1333-1337) using an algorithm that considered side chain interactions.Similarly, the internal features of proteins for design criteria havebeen considered. For example, Dahiyat and Mayo (1997) “Probing the Roleof Packing Specificity in Protein Design” Proc. Natl. Acad. Sci. USA 94:10172-10177, used a protein design algorithm (using a dead-endelimination theorem) that quantitatively considered side-chain packingto consider steric constraints on protein design. Penalization forexposed hydrophobic surface structures also improved design performance.More recently, pairwise expressions with one scalable parameter havebeen used that reproduce true buried and true solvent-accessible areas(Street and Mayo (1998) “Pairwise Calculation of ProteinSolvent-Accessible Surface Areas” Folding & Design 3: 253-258).

As applied to the present invention, steric and other constraints can beused to consider structural linkages between amino acids (and in proteinstructures generally) and these linkages can be used to set filteringcriteria for any GO or GA of interest, e.g., to select appropriatecross-over points between sequences of interest. As has already beenmentioned, a feature of the invention optionally uses recursive cyclesof design and experimentation, with observed results being used toimprove both the activity of any protein of interest and the modelingtools used to predict cross-overs and to perform other GOs.

While amino acid linkages, motif maintenance and other structural/designcriteria provide one basis for selecting cross-over points andperforming other GOs, other approaches such as automated sequenceselection can also be used. For example, in Dahiyat et al. (1997) “DeNovo Protein Design: Towards Fully Automated Sequence Selection” J. Mol.Biol. 273:789-796, a quantitative design algorithm using a combinedexperimental and computational (e.g., the dead-end elimination theorem)approach was used to generate a ∃∃α motif which had 20 out of 28residues determined by modeling. Similarly, in Dahiyat and Mayo (1997)“De Novo Protein Design: Fully Automated Sequence Selection” Science278:82-87 a novel sequence for an entire protein having a ∃∃α structurewas produced. Thus, in the context of the present invention, automatedsequence selection and quantitative protein design is used both toprovide sequences for in silico manipulation and recombination (i.e., asprovided by any design process) and to make structure or activitypredictions that can be used to modulate cross-over selection and otherGOs, e.g., in silico. Again, these processes can include statistical aswell as structure modeling approaches, in any combination, to providecomplex cycles of design, testing and prediction. Essentially anylinkage between structural elements, activity and statistically derivedparameters can be maintained during cycles of in silico operations andphysical recombination and/or selection.

As described by Malakauskas and Mayo (1998) “Design, Structure, andStability of a Hyperthermophilic Protein Variant” Nature Struct. Biol.5:470, design cycles can be used to improve protein activity as well. Inthis example, the stability of a thermophilic protein was increased byselecting residues for mutation based upon design algorithms. Thecombinatorial algorithm that was used simultaneously accounted forcomplex contributions of helix dipole interactions, improvement ofsecondary structure propensity, optimized core packing, increased burialof hydrophobic residues, and the like. In the context of the presentinvention, such combinatorial algorithms can be used to select desiredimprovements, e.g., based upon the functions of the algorithms and thesedesired improvements can be incorporated into one or more in silicosequence string to be recombined with any other sequence string,physically or by in silico methods. For example, residues which thealgorithm indicates as targets for mutation are modified in silico, andcross-overs between the resulting modified sequence(s) and any othersequence(s) are designed to maintain or disrupt the modifications in anysubsequent recombination steps.

An example of protein modification to maintain activity using a modifiedstructure is found in Strop and Mayo (1999) “Rubredoxin Variant FoldsWithout Iron” J. Am. Chem. Soc. 121(11):2341-2345. As described, ironbinding was eliminated from PFRD, while maintaining folding (includingre-folding) and activity. One feature of the present invention is themodeling of proteins to perform such modifications, followed by insilico GOs and/or physical recombination to provide shuffled variants ofproteins which are modeled to have particular activities. In addition tobeing valuable for the production of new proteins of interest, thisrecursive cycling process can be used as a learning heuristic to improvethe predictive value of the modeling system.

Many design methods rely generally on energy expressions to evaluate thequality of different amino acid sequences for target protein structures(reviewed, e.g., in Gordon et al. (1999) “Energy Functions for ProteinDesign” Current Opinion in Structural Biology 9(4):509-513). Forexample, force fields tailored to protein design have been used, e.g.,which consider van der Waals, packing specificity, hydrogen bonding,electrostatics, internal coordinate terms, salvation, entropy and thelike, e.g., in an essentially simultaneous fashion (See also, Gordon etal., id and Malakauskas and Mayo (1998) “Design, Structure, andStability of a Hyperthermophilic Protein Variant” Nature Struct. Biol.5:470). Methods of implementing these algorithms in a combinatorialfashion (e.g., “branch and terminate” algorithms, “dead end elimination”algorithms and other approaches in the references herein) have beendescribed (Gordon and Mayo (1999) “Branch-and-Terminate: A CombinatorialOptimization Algorithm for Protein Design” Structure with Folding andDesign 7(9):1089-1098; Gordon and Mayo (1998) “Radical PerformanceEnhancements for Combinatorial Optimization Algorithms based on theDead-End Elimination Theorem” J. Comp. Chem 19:1505-1514). In general,these methods can be used to bias or modulate any GO (e.g., cross-overselection) to maintain or alter any such force field or, more simply, tomaintain or alter residue any sequence which the algorithms indicate tobe of interest. That is, residues which the algorithm(s) indicates astargets for maintenance or mutation are modified or maintained insilico, as noted herein. Cross-overs between the resulting sequence(s)and any other sequence(s), or any other GO of interest, are designed tomaintain or to disrupt the sequences of interest in any subsequentrecombination step(s).

In any case, designed or modified proteins or character stringscorresponding to proteins can be directly shuffled in silico, or, e.g.,reverse translated and shuffled in silico and/or by physical shuffling(that is, many design algorithms apply primarily to proteins, whilerecombination is conveniently performed between coding nucleicacids—though as noted herein, direct recombination between proteins,particularly in silico, can also be performed). Thus, one aspect of theinvention is the coupling of high-throughput rational design and insilico or physical shuffling and screening of genes to produceactivities of interest. It is possible, using the present methods, tocouple high-throughput rational design and random or semi-randomrecombination methods.

Similarly, molecular dynamic simulations such as those above and, e.g.,Ornstein et al. (emsl.pnl.gov:2080/homes/tms/bms.html (on the world wideweb); Curr Opin Struct Biol (1999) 9(4):509-13) provide for “rational”enzyme redesign by biomolecular modeling & simulation to find newenzymatic forms that would otherwise have a low probability of evolvingbiologically. For example, rational redesign of p450 cytochromes andalkane dehalogenase enzymes are a target of current rational designefforts. Any rationally designed protein (e.g., new p450 homologues ornew alkaline dehydrogenase proteins) can be evolved by reversetranslation and shuffling against either other designed proteins oragainst related natural homologous enzymes. Details on p450s can befound in Ortiz de Montellano (ed.) 1995, Cytochrome P450 Structure andMechanism and Biochemistry, Second Edition Plenum Press (New York andLondon). Furthermore, the dynamic simulations can be used as predictorsof residues of interest and coupling or linkages between residues ofinterest and cross-over selection (or other GOs) can be performed tospecifically maintain or eliminate such linkages in silico.

In addition to the above approaches, homology modeling can also be usedto provide structural predictors and to identify which residues arerelevant to activity. See, Haney et al. (1997) “Structural basis forthermostability and identification of potential active site residues foradenylate kinases from the archaeal genus Methanococcus” Proteins28(1):117-30. As applied to the present invention, this combination ofstructure and sequence analysis can be used to identify putativerelationships between amino acids in a structure and can, accordingly,be used in cycles of design, in silico GO application, recombination,etc. Here again, cross-over selection or other GOs of interest can bemaintained or eliminated in silico.

In addition to utilizing complex design algorithms, any structuralinformation can be used to select cross-over sequences between nucleicacids (or to perform any other GO of interest). For example, comparisonof protein crystal structures to predict crossover points based onstructural rather than sequence homology considerations can be conductedand crossover can be effected by oligos to direct chimerization asdiscussed herein. This can be performed with or without the use ofprotein design algorithms, i.e. even simple inspection of crystalstructure can provide a basis for selecting or eliminating particularresidues. Thus, with knowledge about structure, either complex designalgorithms or simple structural analysis can be used to select residues,secondary structures, tertiary structures, or the like, to be maintainedor disrupted in any recombined coding nucleic acids.

Multivariate Sequence-Activity Modeling of Proteins, Optimization ofEnzymatic Activity by Rational Statistics.

As noted, statistical considerations can be applied to provide any of avariety of filters for identifying relationships between nucleic acid orprotein sequences. This section describes how to analyze a large numberof related protein sequences using modern statistical tools and how toderive novel protein sequences with desirable features using rationalstatistics and multivariate analysis.

Background Multivariate Data Analysis

Multivariate data analysis and experimental design is widely applied inindustry, government and research centers. It is typically used forthings like formulating gasoline, or optimizing a chemical process. Inthe classic example of gasoline formulation, there may be more than 25different additives that can be added in different amounts and indifferent combinations. The output of the final product is alsomultifactorial (energy level, degree of pollution, stability etc. etc.).By using experimental design, a limited number of test formulations canbe made where the presence and amounts of all additives are altered in anon-random fashion in order to maximally explore the relevant“formulation space.” The appropriate measurements of the differentformulations are subsequently analyzed. By plotting the datapoints in amultivariate (multidimensional) fashion, the formulation space can begraphically envisioned and the ideal combination of additives can beextracted. One of the most commonly used statistical tools for this typeof analysis is Principal Component Analysis (PCA).

In this example, this type of matrix is used to correlate eachmultidimensional datapoint with a specific output vector in order toidentify the relationship between a matrix of dependant variables Y anda matrix of predictor variables X. A common analytical tool for thistype of analysis is Partial Least Square Projections to LatentStructures (PLS). This, for instance, is often used in investmentbanker's analysis of fluctuating stock prices, or in material sciencepredictions of properties of novel compounds. Each datapoint can consistof hundreds of different parameters that are plotted against each otherin an n-dimensional hyperspace (one dimension for each parameter).Manipulations are done in a computer system, which adds whatever numberof dimensions are needed to be able to handle the input data. There arepreviously mentioned methods (PCA, PLS and others) that can assist infinding projections and planes so that hyperspace can be properlyanalyzed.

Background Sequence Analysis

Nucleotide or amino acid sequence analysis has traditionally beenconcentrated on qualitative pattern recognition (e.g., sequenceclassification). This mainly involves identifying sequences based onsimilarity. This works well for predictions or identifications ofclassification, but does not always correlate with quantitative values.For instance, a consensus transcriptional promoter may not be a goodpromoter in a particular application, but is, instead, the averagepromoter among an aligned group of related sequences.

To access the quantitative features of related biological sequences(DNA/RNA or amino acids) one can analyze the systematic variations (i.e.systematic absence of similarity) among aligned sequences with relatedbiological activity. By applying different multivariate analysis tools(such as PLS) to protein sequences, it is possible to predict a sequencethat generates better catalytic activity than the best one present inthe analyzed set. Experimental data showing the success of the generalmethod is described below.

Background Promoter Activity Multivariate Analysis

One of the few references where multivariate data analysis has beenapplied to biological sequences was focused on analyzing a set ofdefined transcriptional promoters in order to see if one could predict astronger promoter than any of those found in the training set (Jonssonet al. (1993) Nucleic Acids Res. 21:733-739).

In this example, promoter sequences were parametrizised. For simplicity,the physical-chemical differences between the respective nucleotides(A,C,G,T) were selected as equal, i.e. no nucleotide was considered moreclosely related to any other nucleotide. They were represented as fourdiametrically opposed corners of a cube forming a perfect tetrahedron.By assigning an origin to the center, each corner can be represented bya numerical coordinate, reflecting the numeric representation. Since thedescriptors solely represents equal distribution of properties, anynucleoside can be positioned at any corner. See, FIG. 17.

Previous studies (Brunner and Bujard (1987) EMBO J. 6; 3139-3144; Knausand Bujard (1988) EMBO J. 7:2919-2923; Lanzer and Bujard (1988) Proc.Natl. Acad. Sci. 85:8973-8977) analyzed a set of 28 promoters that hadbeen studied in detail in the identical context. The promoters includedE. coli promoters, T5 phage promoters and a number of chimeric andsynthetic promoters. They were all cloned as 68 bases (−49 to +19)inserted in front of a DHFR coding region. Relative transcriptionallevel was measured by dot-blot using the vector derived β-lactamase geneas internal standard.

In this example, when the 28 promoters, each with 68 nucleotides, wereparameterized with the three descriptors as defined in FIG. 14, theresult is a 28×204 matrix (28 promoters×68 nucleotides×3 parameters(steric bulk, hydrophobicity and polarizability)). The unique sequenceof each promoter can be represented as a single point in a204-dimensional hyperspace. This compilation of 28 promoters thus formeda cluster of 28 points in this space. The experimental data fromprevious studies noted above was repeated and the generatedtranscription levels plotted against the cluster of 28 points generatedin the 204 dimensional hyperspace using PLS. Half of the promoters (14)were subsequently used to build a statistical model and the other halfto test it. This was showed to generate a good correlation forcalculated vs. observed promoter strength.

Two new promoters were constructed based on extrapolations of thegenerated model and in both cases were shown to be significantly betterthan the best promoter present among the 28 initial promoters.

Multivariate Analysis And Protein Sequence

The same analytical methods can be applied to protein sequences. Signalpeptides have been characterized using multivariate analysis showinggood correlation between location in hyperspace and final physicallocalization (Sjöström et al. (1987) EMBO J. 6:823-831). The maindifference between nucleotides and amino acids being that instead ofqualitative descriptors of the nucleotides (see FIG. 14), quantitativedescriptors have to be used to parameterize amino acids. The relevantfeatures of the amino acids (steric bulk, hydrophobicity andpolarizability) have been determined and can be extracted from theliterature (Hellberg et al. (1986) Acta Chem. Scand. B40: 135-140;Jonsson et al., (1989) Quant. Struct.—Act. Relat. 8:204-209).

Following shuffling and characterization of shuffled proteins, themutated proteins are analyzed, both those that are “better” and thosethat are “worse” than the initial sequences. Statistical tools (such asPLS) can be used to extrapolate hovel sequences that are likely to bebetter than the best sequence present in the analyzed set. Cross-overscan be selected (or other GOs performed) that provide for generation ofthe extrapolated sequences.

Modeling Protein Sequence Space

As described above, any protein encoded by a DNA sequence can be plottedas a distinct point in multidimensional space using statistical tools. A“normal” 1 kb gene can constitute e.g., about 330 amino acids. Eachamino acid can be described by, e.g., three major physico-chemicalquantitative descriptors (steric bulk, hydrophobicity andpolarizability) for each amino acid (other descriptors for proteins arelargely dependent on these three major descriptors). See also, Jonssonet al. (1989) Quant. Struct.—Act. Relat. 8:204-209. Thus, a 1 kb gene ismodeled in 330 (number of amino acids)×20 (possible amino acids at eachposition)×3 (the three main descriptors noted above, for each aminoacid)=19,800 dimensions. Because of the extended nature of sequencespace, a number of shuffled sequences are used to validate sequenceactivity related predictions. The closer the surrounding sequences arein space (percent similarity), the higher the likelihood that predictivevalue can be extracted. Alternatively, the more sequence space which isanalyzed, the more accurate predictions become. This modeling strategycan be applied to any available sequence. As described above, cycles ofdesign and experimentation can be used to refine the model.

Alternatively, neural networks can be used to learn a type of patternand predict the generated outcome of given variations. Examples of suchneural networks include Schneider and Wrede (1998) “Artificial neuralnetworks for computer-based molecular design” Prog Biophys Mol Biol1998; 70(3): 175-222; Schneider et al. (1998) “Peptide design byartificial neural networks and computer-based evolutionary search” ProcNatl Acad Sci USA 95(21):12179-84; and Wrede et al. (1998) “Peptidedesign aided by neural networks: biological activity of artificialsignal peptidase I cleavage sites” Biochemistry 37(11):3588-93.

The prognostic value of plotting a large number of shuffled sequences isdescribed above. Two additional approaches can also be used. First,plotting as many chimeric progeny in a library as possible vs. anenzymatic activity using, for example, PLS (partial least squareprojections to latent structures) can be performed. If enough data isavailable, the sequence-activity plot forms a function that can beextrapolated outside of the experimental data to produce an in-silicosequence corresponding to an activity higher than the best training set.Second, all related sequences can be plotted and certain sequencesgrouped with given related activities. Using this matrix, subsequentgenes can immediately be grouped with appropriate activity or newrelated activities can be directly screened for by a subset of thesequenced clones. Cross-overs can be selected to produce any desiredrecombinants generated by the set.

An overall issue for the above strategy is the availability of enoughrelated sequences generated through shuffling to provide usefulinformation. An alternative to shuffling sequences is to apply themodeling tools to all available sequences, e.g., the GenBank databaseand other public sources. Although this entails massive computationalpower, current technologies make the approach feasible. Mapping allavailable sequences provides an indication of sequence space regions ofinterest. In addition, the information can be used as a filter which isapplied to in silico shuffling events to determine which virtual progenyare preferred candidates for physical implementation (e.g., synthesisand/or recombination as noted herein). Thus, cross-overs and other GOsare selected to provide preferred recombinants and/or substrates forshuffling.

Identifying Crossover Sites for Synthetic Shuffling

As discussed herein and, e.g., in “OLIGONUCLEOTIDE MEDIATED NUCLEIC ACIDRECOMBINATION” by Crameri et al., Filed Jan. 18, 2000, U.S. Ser. No.09/484,850, Synthetic shuffling optionally uses bridging oligos to forcecrossovers at defined location(s) in order to generate chimeric progeny.The general concept of defining the location for forced crossover oncertain primary, secondary and tertiary sequence based considerations isdiscussed herein. Alternatively statistical matrixes such as Markovchains can be used to pinpoint ideal crossover locations. This examplepresents two alternative criteria for defining crossover locations basedon statistical considerations.

Co-variation of amino acids during evolution allows proteins to retain agiven fold or function while altering other traits, such as specificity.The co-variation identified in a large dataset can be useful inaddressing possible crossover locations as it identifies co-evolvingamino acids in a given family and allows bridging oligos to beengineered so that such functional constraints are retained.

The difficulty with this type of analysis has been that the signal fromstructurally/functionally constrained amino acids are masked by a muchlarger set of amino acids that show covariance due to phylogeneticrelationship. Separating the functional from the phylogenetic covariancehas been typically performed by consideration of high quality crystalstructure information. However, other approaches are feasible.

Approach 1

For example, as described by Wollenberg and Atchley (2000) Proc. Natl.Acad. Sci. USA 97; 3288-3291, a statistical approach is used to separatephylogenetic association vs. functional association of a given aminoacid covariance. This approach uses a parametric bootstrap algorithm toconstruct a statistical distribution that reflects the associationbetween amino acids exclusively resulting from common ancestry (andchance) and subtracts those positions from all the positions showingcovariance.

Intersite association is determined using Mutual Information (MI)statistics (Shannon & Weaver (1949) The mathematical theory ofinformation. Univ. Illinois Press, Urbana, Ill.; Applebaum (1996)Probability and information: An integrated approach Cambridge Univ.Press, New York). MI measures the extent of association between twopositions in a sequence beyond that expected from chance. The mutualinformation MIXY between sites X and Y is calculated as:

${{MI}_{XY} = {\sum\limits_{i}{\sum\limits_{j}{{P\left( {X_{i},Y_{j}} \right)}\log_{n}\frac{P\left( {X_{i},Y_{j}} \right)}{{P\left( X_{i} \right)}{P\left( Y_{j} \right)}}}}}},$

where P(X_(i)) is the probability of I at site X, P(Y_(j)) is theprobability of j at site Y, and P(X_(i), Y_(i)) is the joint probabilityof i at site X and j at site Y (X≠Y). The maximum MI value occurs whenthe variation at two sites is perfectly correlated. The data presentedby Wollenberg and Atchley uses the sequence elements as symbol variableswith no underlying metric. The same statistical correlation can be doneusing the principal components for each amino acid. Replacing thesymbols with true physicochemical properties can improve thesignificance of the MI relationship.

Setting a statistical acceptability threshold permits theidentification, within a quantifiable error, of those intersiteassociations most probably arising from structural/functional causes.The reminder of the intersite associations can be deemed originatingfrom phylogenetic resemblance. Cross-overs or other GOs are selected toproduce the intersite associations of interest.

Approach Two

Pollock et al. (1999) J. Mol. Biol. 287:187-198 present a maximumlikelihood method to identify coevolving protein residues. The data isreduced to a two-state system at each site using size and charge asamino acids characteristics. This reduces the complexity ofcoevolutionary relationships and aids in identifying the primarycomponent of pairwise coevolution. The same method can be used with theprincipal components of each amino acid, ensuring a gradual nominationof entities, instead of simple binary nomination, resulting insignificantly higher resolution and lower noise levels.

In short, by binary characterization of each position and comparison ofall of the nodes in a matrix of all amino acids in all proteins presentin the homology-cluster, correlations are scored (maximum likelihood)and related to the phylogentic dendrogram generated for the samecluster. A covariance correlation can be used to identify which residuesare functionally/structurally linked. See also, Pollock et al. (1999),id. Cross-overs are selected to maintain these relationships.

Shuffling of Cladistic Intermediates

The present invention provides for the shuffling of “evolutionaryintermediates.” In the context of the present invention, evolutionaryintermediates are artificial constructs which are intermediate incharacter between two or more homologous sequences, e.g., when thesequences are grouped into an evolutionary dendrogram.

Nucleic acids are often classified into evolutionary dendrograms (or“trees”) showing evolutionary branch points and, optionally,relatedness. For example, cladistic analysis is a classification methodin which organisms or traits (including nucleic acid or polypeptidesequences) are ordered and ranked on a basis that reflects origin from apostulated common ancestor (an intermediate form of the divergent traitsor organisms). Cladistic analysis is primarily concerned with thebranching of relatedness trees (or “dendrograms”) which showsrelatedness, although the degree of difference can also be assessed (adistinction is sometimes made between evolutionary taxomomists whoconsider degrees of difference and those who simply determine branchpoints in an evolutionary dendrogram (classical cladistic analysis); forpurposes of the present invention, however, relatedness trees producedby either method can produce evolutionary intermediates).

Cladistic or other evolutionary intermediates can be determined byselecting nucleic acids which are intermediate in sequence between twoor more extant nucleic acids. Although the sequence may not exist innature, it still represents a sequence which is similar to a sequence innature which had been selected for, i.e., an intermediate of two or moresequences represents a sequence similar to the postulated commonancestor of the two or more extant nucleic acids. Thus, evolutionaryinter-mediates are one preferred shuffling substrate, as they represent“pseudo selected” sequences, which are more likely than randomlyselected sequences to have activity.

One benefit of using evolutionary intermediates as substrates forshuffling (or of using oligonucleotides which correspond to suchsequences) is that considerable sequence diversity can be represented infewer starting substrates (i.e., if starting with parents A and B, asingle intermediate “C” has at least a partial representation of both Aand B). This simplifies the oligonucleotide synthesis scheme for genereconstruction/recombination methods, improving the efficiency of theprocedure. Further, searching sequence databases with evolutionaryintermediates increases the chances of identifying related nucleic acidsusing standard search programs such as BLAST.

Intermediate sequences can also be selected between two or moresynthetic sequences which are not represented in nature, simply bystarting from two synthetic sequences. Such synthetic sequences caninclude evolutionary intermediates, proposed gene sequences, or othersequences of interest that are related by sequence. These “artificialintermediates” are also useful in reducing the complexity of genereconstruction methods and for improving the ability to searchevolutionary databases.

Accordingly, in one significant embodiment of the invention, characterstrings representing evolutionary or artificial inter-mediates are firstdetermined using alignment and sequence relationship software and thensynthesized using oligonucleotide reconstruction methods. Alternately,the intermediates can form the basis for selection of oligonucleotidesused in the gene reconstruction methods herein.

Several of the following sections describe implementations of thisapproach using hidden Markov model threading and other approaches.

In Silico Shuffling Using Hidden Markov Model Threading

A concern with synthetic shuffling is the assumption that each aminoacid present among the parents is an independent entity and add (or doesnot add) function in a given functional dimension by itself. Whenshuffling using DNAse I based methods, this problem is avoided becauserecombination during assembly occurs as 20-200 bp fragments and, thus,each amino acid exists in its evolutionary context among other aminoacids that have co-evolved in a given direction due to selectivepressure of the functional unit (gene or promoter or other biologicalentity). By capturing the co-variance normally existing within a familyof genes, either wild type or generated through regular shuffling, asignificant number of biologically inactive progeny are avoided,improving the quality of the generated library. Artificially generatedprogeny may be inactive due to structural, modular or other subtleinconsistencies between the active parents and the progeny.

One way of weeding out unwanted non co-variance progeny is to apply astatistical profile such as Hidden Markov Model (HMM) on the parentalsequences. An HMM matrix generated (e.g., as in FIG. 15) can capture thecomplete variation among the family as probabilities between allpossible states (i.e. all possible combinations of amino acids,deletions and insertion). The matrix resulting from the analyzed familyis used to search assorted databases for additional members of thefamily that are not similar enough to be identified by standard BLASTalgorithms of any one particular sequence, but which are similar enoughto be identified when probing using a probabilistic distribution patternbased on the original family.

The HMM matrix shown in FIG. 15 exemplifies a family of 8 amino acidpeptides. In each position, the peptide can be a specific amino acid(one of the 20 present in the boxes), an insertion (diamonds), or adeletion (circles). The probability for each to occur is dependent onhow often it occurs among the compiled parents. Any given parent cansubsequently be ‘threaded’ through the profile in such way that allallowed paths are given a probability factor.

HMM can be used in other ways as well. Instead of applying the generatedprofile to identify previously unidentified family members, the HMMprofile can be used as a template to generate de novo family members(e.g., intermediate members of a cladistic tree of nucleic acids). Forexample, the program, HMMER is available (hmmer.wustl.edu/, on the worldwide web). This program builds a HMM profile on a defined set of familymembers. A sub-program, HMMEMIT, reads the profile and constructs denovo sequences based on that. The original purpose of HMMEMIT is togenerate positive controls for the search pattern, but the program canbe adapted to the present invention by using the output as in silicogenerated progeny of a HMM profile defined shuffling. According to thepresent invention, oligonucleotides corresponding to these nucleic acidsare generated for recombination, gene reconstruction and screening.

As the sequence context of each position is accounted for in aprobabilistic fashion, the number of non-active progeny is significantlylower than a shuffling reaction that simply randomly selects suchprogeny. Crossover between genetic modules (structural, functional, ornon-defined) occur where they occur in nature (i.e. among the parents)and co-evolution of point mutations or structural elements is retainedthroughout the shuffling process.

Example Algorithm for Generating Sequence Intermediates from SequenceAlignments

The following is an outline for a program for generating sequenceintermediates from alignments of related parental nucleic acids.

Given an alignment of sequences which code for the parents, and analignment which codes for the children for each child sequence  for eachparent sequence   for each window    if the parent sequence and childsequence match for this window     If this window not already covered bysequences in segment list      Try to expand window, 5′ and 3′ until toomany mismatches      Add final expanded segment to segment list for thissequence for each child sequence  set position to start of sequence  dothe following until the end of the child sequence is reached   Searchthrough segments finding a segment that extends the longest   from apoint before position (this is most like the parent segment)   If one isfound add to optimal path list and set position to end of   foundsegment   if one is not found increment current position displaysegments from optimal path listNormalization of Libraries—Use of Positive or Negative Activity Data

One aspect of the present invention is to use positive or negative datain sequence design and selection methods, either in silico, or inphysical processing steps, or both. The use of positive or negative datacan be in the context of a learning heuristic, a neural network or bysimply using positive or negative data to provide logical or physicalfilters in design and library synthesis processes. Learning networks aredescribed supra, and provide one convenient way of using positive ornegative data to increase the chances that additional sequences whichare subsequently generated will have a desired activity. The ability touse negative data to reduce the size of libraries to be screenedprovides a considerable advantage, as screening is often a limiting stepin generating improved genes and proteins by forced evolution methods.Similarly, the use of positive data to bias libraries towards sequencesof interest is another way of focusing libraries.

For example, as noted, in addition to a neural net learning approach,positive or negative data can be used to provide a physical or logical“filter” for any system of interest. That is, sequences which are shownto be inactive provide useful information about the likelihood thatclosely related sequences will also prove to be inactive, particularlywhere active sequences are also identified. Similarly, sequences whichare active provide useful information about the likelihood that closelyrelated sequences will also prove to be active, particularly whereinactive sequences are also identified. These active or inactivesequences can be used to provide a virtual or physical filter to biaslibraries (physical or virtual) toward production of more activemembers.

For example, when using negative data, physical subtraction methods usehybridization to inactive members under selected stringency conditions(often high stringency, as many libraries produced by the methods hereincomprise homologous members) to remove similar nucleic acids fromlibraries which are generated. Similarly, hybridization rules or otherparameters can be used to select against members that are likely to besimilar to inactive sequences. For example, oligonucleotides used ingene reconstruction methods can be biased against sequences which havebeen shown to be inactive. Thus, in certain methods, libraries orcharacter strings are filtered by subtracting the library or set ofcharacter strings with members of an initial library of biologicalpolymers which display activity below a desired threshold.

When using positive data, physical enrichment methods use hybridizationto active members under selected stringency conditions (often highstringency, as many libraries produced by the methods herein comprisehomologous members) to isolate similar nucleic acids which comprise themembers of libraries to be produced. Similarly, hybridization rules orother parameters can be used to select for members that are likely to besimilar to active sequences. For example, oligonucleotides used in genereconstruction methods can be biased towards sequences which have beenshown to be active. Thus, in certain methods, libraries or characterstrings are filtered by biasing the library or set of character stringswith members of an initial library of biological polymers which displayactivity above a desired threshold.

Similarly, in silico approaches can be used to produce libraries ofinactive sequences, rather than active sequences. That is, inactivesequences can be shuffled in silico to produce libraries of clones thatare less likely to be active. These inactive sequences can be physicallygenerated and used to subtract libraries (typically throughhybridization to library members) generated by other methods. Thissubtraction reduces the size of the library to be screened, primarilythrough the elimination of members that are likely to be inactive.

Example—Motif Filtering

Selections or screens often yield too many “positive” clones tocost-effectively sequence all of the positive or negative clones.However, if sequence motifs are identified that are enriched ordepopulated in either the positive or the negative clones, then thisbias is used in the construction of synthetic libraries that are biasedtoward “good” motifs and biased away from “bad” motifs.

If each contiguous selected region or motif (e.g., the selected windowcan be, e.g., a 20 base region) is thought of as a separate gene or geneelement, one can measure the change in gene frequencies before and aftera selection or screen. Motifs that increase in frequency in the positiveclones are characterized as “good”, and motifs whose frequency isreduced in the positive clones are characterized as “bad.” Secondgeneration libraries are synthesized in which the library is selected tobe enriched for good motifs and depopulated with bad motifs, using anyfiltering or learning process as set forth herein.

A variety of methods for measuring frequencies of motifs in populationsof genes are available. For example, one can hybridize analyte sequencesto a gene chip or other array of nucleic acids with the motifs ofinterest encoded in a spatially addressable fashion, e.g., using genechips as provided by Affymetrix (Santa Clara, Calif.), or other genechip manufacturers. Similarly, hybridization to membranes containingspatially addressable motifs and measuring relative signal intensitiesfor probe before and after selection can also be performed in anessentially similar fashion, e.g., using standard Southern or northernblot methods. Relative ratios of identified desirable/undesirablefeatures on the chips also provides an indication of overall libraryquality. Similarly, phage display or other expression libraries can beused to assess library features, i.e., by evaluating expressionproducts.

Alternatively, real time quantitative PCR (e.g., TAQMAN™ PCR Reagents)can be performed where PCR oligos are highly discriminating for thefeature of interest. This can be done by, for example, having apolymorphism unique to the motif present at or near the 3′ end of anoligo such that it will only prime the PCR efficiently if there is aperfect match. Real time PCR product analysis by, e.g., FRET or TAQMAN™PCR Reagents (and related real time reverse-transcription PCR) is afamily of known techniques for real time PCR monitoring that has beenused in a variety of contexts (see, Laurendeau et al. (1999) “TaqManPCR-based gene dosage assay for predictive testing in individuals from acancer family with INK4 locus haploinsufficiency” Clin Chem 45(7):982-6;Laurendeau et al. (1999) “Quantitation of MYC gene expression insporadic breast tumors with a real-time reverse transcription-PCR assay”Clin Chem 59(12):2759-65; and Kreuzer et al. (1999) “LightCyclertechnology for the quantitation of bcr/ab1 fusion transcripts” CancerResearch 59(13):3171-4.

If the gene family of interest is highly similar to begin with (forexample, over 90% sequence identity), then one can simply sequence thepopulation of genes before and after selection. If several sequencingprimers up and down the gene are used, then one can look at thesequences in parallel on a sequencing gel. The sequence polymorphismsnear the primer can be read out to see the relative ratio of bases atany given site. For example, if the population starts out with 50% T and50% C at a given position, but 90% T and 10% after selection, one couldeasily quantitate this base ratio from a sequencing run that originatesnear the polymorphism. This method is limited, because as one getsfurther from the primer and reads through more polymorphisms, themobilities of the various sequences gets increasingly variable and thetraces begin to run together. However, as the cost of sequencingcontinues to decline and the cost of oligos continues to decrease, onesolution is simply to sequence with many different oligos up and downthe gene.

Example: Fractional Distillation of Sequence Space

Typical sequence spaces are very large compared to the number ofsequences that can be physically cloned and characterized. Computationaltools exist with which to describe subsets of sequence space which arepredicted to be enriched for clones with properties of interest (see,supra). However, there are assumptions and computational limitationsinherent to these models. Methods for fractionating sequence space suchthat it is enriched for molecules which are predicted by a given modelto have greater or lesser fitness with respect to a phenotype ofinterest would be useful for testing such predictive models.

A simple example of how this would work is as follows. There are about10²⁷ possible shuffled IFNs (a fairly typical protein in terms of size)based on the naturally occurring human IFN gene family. This is largerthan the number that can be easily screened. If one's goal is to evolveshuffled human IFNs that are active, e.g., on mouse cells, then onecould use the information in the literature that shows that residues 121and 125 from human IFN alpha I confer improved activity whentransplanted onto other human II-Ns such as IFN-∀2a. If one assumes thatthis motif confers improved activity in many different contexts, thenone can create a large pool of shuffled IFN genes (typically on theorder of 10⁹-10¹²), convert them to ssDNA, pass them over an affinitycolumn consisting of an oligonucleotide complementary human IFN alpha1over these residues, wash under appropriate stringency, elute the boundmolecules, PCR amplify the eluted genes, clone the material, and performfunctional tests on the expressed clones. This protocol allows one tophysically bias a library of shuffled genes strongly in favor ofcontaining this motif which is predicted by this very simple model toconfer an improvement in the desired activity.

Ideally, one takes populations that are enriched for the motif andpopulations that are depopulated with the motif. Both populations areanalyzed (say 1000 clones from each population). The hypothesis is then“tested” asking whether this fractionation of sequence space biased theaverage fitness in the predicted way. If it did, then one could “accept”the hypothesis and scale up the screen of that library. One could alsotest a number of design algorithms by affinity based fractionation,accept the ones that are supported by the results of the experiment, andthen perform the affinity selections in series so that one enriches forclones that meet the design criteria of multiple algorithms.

In this model, shuffling, such as family shuffling, is used as the firstorder design algorithm. However, additional design algorithms areintegrated downstream of the shuffling to further fractionate a sequencespace based on simple design heuristics. The method can be performed atthe nucleic acid level with any design algorithm that can be translatedinto a nucleic acid selection scheme.

A number of variations on this example are useful for reducing the sizeof libraries that are produced by physical or virtual filteringprocesses.

For example, affinity electing oligonucleotides that encode motifs ofinterest prior to gene recombination/resynthesis (either physically orin silico) reduces the diversity of populations of nucleic acids thatare produced in gene recombination/resynthesis methods as noted herein.

Similarly, oligonucleotides encoding motifs can be selected byenzymatically degrading molecules that are not perfectly matched withthe oligos, e.g., again prior to gene recombination/resynthesis methods.Alternately, genes that match imperfectly with the oligonucleotides canbe selected for, e.g., by binding to mutS or other DNA mismatch repairproteins.

Polymerization events during recombination/gene synthesis protocols canbe primed using one or more oligos encoding the motif(s) of interest.That is, mismatches at or near the 3′ end of hybridized nucleic acidsreduce or block elongation. In this variation, only newly polymerizedmolecules are allowed to survive (used in subsequent libraryconstruction/selection steps). This can be done, for example, by primingreverse transcription of RNA and then degrading the RNA.

Another approach is to make the template specifically degradable. Forexample, DNA with a high frequency of uracil incorporation can besynthesized. Polymerase-based synthesis is primed with oligos andextended with dNTPs containing no uracil. The resulting products aretreated with uracil glycosylase and a nuclease that cleaves at apurinicsites, and the degraded template removed. Similarly, RNA nucleotides canbe incorporated into DNA chains (synthetically or via enzymaticincorporation); these nucleotides then serve as targets for cleavage viaRNA endonucleases. A variety of other cleavable residues are known,including certain residues which are targets for enzymes or otherresidues and which serve as cleavage points in response to light, heator the like. Where polymerases are currently not available with activitypermitting incorporation of a desired cleavage target, such polymerasescan be produced using shuffling methods to modify the activity ofexisting polymerases, or to acquire new polymerase activities.

Localized motifs can easily be translated into affinity selectionprocedures. However, one sometimes wants to impose a rule that moleculeshave multiple sequence features that are separated in space in the gene(e.g., 2, 3, 4, 5, 6, etc. sequence features). This can be timed into aselection by making a nucleic acid template that contains all motifs ofinterest separated by a flexible linker. The T_(m) for molecules havingall motifs is greater than for molecules having only one or two of themotifs. It is, therefore, possible to enrich for molecules having allmotifs by selecting for molecules with high T_(m)s for the selectingoligo(s).

A “gene” of many such motifs strung together can be synthesizedseparated by flexible linkers or by bases such as inosine that can basepair promiscuously. One would then select for genes with high T_(m)s forthe selecting nucleic acid. Careful design of the selecting nucleic acidtemplate allows one to enrich for genes having a large number ofsequence motifs that are predicted to bias genes containing them towardhaving a phenotype of interest.

If there is little information about whether any given motif ispredicted to favorably bias the library, the technique can still beused. A set of motifs is defined, e.g., based upon sequence conservationbetween different homologs, or the motifs can even be randomly selectedmotifs. As long as the sequence space is not isotropic (equally densewith good members in all directions), then one can simply fractionatethe sequence space based on a designed or on a random set of motifs,measure the average fitness of clones in the region of sequence space ofinterest, and then prospect more heavily in the regions that give thehighest fitness.

In addition to simple sequence alignment methods, there are moresophisticated approaches available for identifying regions of interestsuch as macromolecule binding sites. For example, U.S. Pat. No.5,867,402 to Schneider, et al (1999) “COMPUTATIONAL ANALYSIS OF NUCLEICACID INFORMATION DEFINES BINDING SITES” proposes methods in whichbinding sites are defined based upon the individual information contentof a particular site of interest. Substitutions within the binding sitesequences can be analyzed to determine whether the substitution causes adeleterious mutation or a benign polymorphism. Methods of identifyingnew binding sites using individual information content are alsoproposed. This approach can be used in the context of the presentinvention as one way of identifying sequences of interest for in silicomanipulation of the sequences.

Motif Breeding

Rational design can be used to produce desired motifs in sequences orsequence spaces of interest. However, it is often difficult to predictwhether a given designed motif will be expressed in a functional form,or whether its presence will affect another property of interest. Anexample of this is the process of designing glycosylation sites intoproteins such that they are accessible to cellular glycosylationmachinery and such that they do not negatively affect other propertiesof the protein such as blocking binding to another protein by virtue ofsteric hindrance by the attached polysaccaride groups.

One way of addressing these issues is to design motifs or multiplevariations of motifs into multiple candidate sites within the targetgene. The sequence space is then screened or selected for thephenotype(s) of interest. Molecules that meet the specified designcriterion threshold are shuffled together, recursively, to optimizeproperties of interest.

Motifs can be built into any gene. Exemplary protein motifs include:N-linked glycosylation sites (i.e. Asn-X-Ser), 0-linked glycosylationsites (i.e. Ser or Thr), protease sensitive sites (i.e. cleavage bycollagenase after X in P-X-G-P) Rho-dependent transcriptionaltermination sites for bacteria, RNA secondary structure elements thataffect the efficiency of translation, transcriptional enhancer elements,transcriptional promoter elements, transcriptional silencing motifs,etc.

High Throughput Rational Design

In addition to, or in conjunction with the rational design approachesdescribed supra, high throughput rational design methods are alsouseful. In particular, high throughput rational design methods can beused to modify any given sequence in silico, e.g., beforerecombination/synthesis. For example, Protein Design Automation (PDA) isone computationally driven system for the design and optimization ofproteins and peptides, as well as for the design of proteins andpeptides.

Typically, PDA starts with a protein backbone structure and designs theamino acid sequence to modify the protein's properties, whilemaintaining its three dimensional folding properties. Large numbers ofsequences can be manipulated using PDA, allowing for the design ofprotein structures (sequences, subsequences, etc.). PDA is described ina number of publications, including, e.g., Malakauskas and Mayo (1998)“Design, Structure and Stability of a Hyperthermophilic Protein Variant”Nature Struc. Biol. 5:470; Dahiyat and Mayo (1997) “De Novo ProteinDesign: Fully Automated Sequence Selection” Science, 278, 82-87.DeGrado, (1997) “Proteins from Scratch” Science, 278:80-81; Dahiyat,Sarisky and Mayo (1997) “De Novo Protein Design: Towards Fully AutomatedSequence Selection” J. Mol. Biol. 273:789-796; Dahiyat and Mayo (1997)“Probing the Role of Pachng Specificity in Protein Design” Proc. Natl.Acad. Sci. USA, 94:10172-10177; Hellinga (1997) “Rational ProteinDesign—Combining Theory and Experiment” Proc. Natl. Acad. Sci. USA, 94:10015-10017; Su and Mayo (1997 j″ Coupling Backbone Flexibility andAmino Acid Sequence Selection in Protein Design” Prot. Sci. 6:1701-1707;Dahiyat, Gordon and Mayo (1997) “Automated Design of the SurfacePositions of Protein Helices” Prot. Sci., 6:1333-1337; Dahiyat and Mayo(1996) “Protein Design Automation” Prot. Sci., 5:895-903. Additionaldetails regarding PDA are available, e.g., at xencor.com (on the worldwide web).

In the context of the present invention, PDA and other design methodscan be used to modify sequences in silico, which can besynthesized/recombined in shuffling protocols as set forth herein.Similarly, PDA and other design methods can be used to manipulatenucleic acid sequences derived following selection methods. Thus, designmethods can be used recursively in recursive shuffling processes.

In Silico Shuffling: Computational Methods for Designing Useful BridgingOligonucleotides to Facilitate Low Homology Recombination

Physically shuffling sequences that are less than about 50% identical atthe DNA level often results in a low number of recombination sitesbetween the sequences to be shuffled. A number of approaches to solvingthis problem are described herein, including design and construction of“cross-over” or “bridging” oligonucleotides, the use of intermediate“diplomat” sequences and the like.

In one general approach, as noted herein, one way to increase the numberof crossover sites is to build “bridging” or “cross-over-”oligonucleotides that have some number of bases (e.g., sufficient tomediate hybridization, typically, e.g., about 20 or more, though fewercan also be used) identical to one parent, then some number (typically,e.g., also about 20 or more bases) identical to the second parent.

In the context of the present invention, there are at least four basicways of providing bridging oligos, i.e., 1) arbitrarily choosingbridging oligos; 2) making all possible bridging oligos; 3) making asubset of possible bridging oligonucleotides based upon known featuresof the sequences to be shuffled (e.g., to permit particular domains orsubdomains to be recombined or disrupted, e.g., to account for 3-D orempirically derived considerations), and 4) using computational methodsto optimize bridging oligonucleotide selection and design. This lastoption is especially useful in the context of the present invention,although the first three approaches can also be applicable.

As noted above, computational methods exist to examine a proteinsequence to assess the structural stability of variants, based on eitheran available crystal structure, or energy minimization mapping to thecrystal structure of a homologous sequence (see, e.g., references citedin previous section). Although the number of variants that can bescreened in this way are large (e.g., on the order of 10⁸⁰ or more) thisis still only a very small fraction of the total possible sequence spaceeven for a single small protein (with 20 variants in a 300 amino acidprotein, 10⁸⁰ represents one variant out of every 2³⁰⁰×10²²⁰.) Thesemethods are, therefore, currently limited in the way in which variantsare chosen. Further, because only a fraction of sequence space isanalyzed, variants are often selected to be close to the active site.

For convenience and clarity of illustration, this section uses shufflingof p450 super family members to illustrate certain methods of performingrelevant steps of the invention; however, the steps are universallyapplicable to other systems. For example, the methods are applicable toany set or sets of proteins for which more than one sequence isavailable and for which at least one structure is either known or can becalculated/estimated.

In brief, desirable crossover points can be selected between two or moresequences, e.g., following an approximate sequence alignment, performingMarkov chain modeling, or any other desired selection method. Thisdesirable subset of all possible crossover points is smaller than allpossible crossover points, and all possible recombinants between two ormore low homology sequences using these reasonable crossover points canbe computationally generated. These recombinants are analyzed for theirability to fit one or more of the possible structures that are eitherknown or calculated for one or more of the selected parental sequences.In this way, it is possible to identify particularly productivecrossover points, and thus to again reduce the total number of bridgingoligonucleotides, this time to a number which can actually besynthesized to provide a useful number of bridging oligonucleotides tofacilitate low homology shuffling reactions. This bridgingoligonucleotide selection method also greatly expands the effectivesequence space that can be analysed computationally.

Description of the Basic Method

In summary, the following steps are performed in bridgingoligonucleotide design and selection, as noted above.

1. Identify proteins from which chimeric recombinants are desirable.These optionally encode actual or putative homologous structures.

(Steps 2-5 below are optionally omitted and only bridgingoligonucleotides fulfilling given criteria (retaining certainsubstructures, statistical considerations, etc.) are used for subsequentlibrary generation).

2. Computationally generate all possible single crossover events betweeneach pair of sequences (or a pre-selected subset of these, e.g.,designed to provide crossover points in regions of low, but existing,homology). For two 1000 amino acid proteins, all possible cross-overpoints is, roughly, 2×1000×1000 crossover points (2×10⁶) (the number canvary somewhat, depending on how the sequences are aligned). For ten 1000amino acid proteins this number is 45 (number of possible pairwisecombinations)×2×1000×1000 (9×10⁷).

3. Calculate the structures of all parents (“template structures”),either from direct measurement (i.e., crystallography, NMR etc.), or byenergy minimization to the known structure of a related protein. Asnoted above, PDA is one particularly useful computationally drivensystem for the design and optimization of proteins and peptides, as wellas for the design of proteins and peptides.

Many other energy minimization and other protein/nucleic acid designalgorithms are known and available. These include computer searchalgorithms in protein modification and design (Desjarlais and Clarke(1999) Structure Fold Des; 7(9); 1089-98), Theoretical and algorithmicaloptimization of the dead-end elimination theorem (Desmet et al. (1997)Pac Symp Biocomput 122-33), fast conformational search strategies forfinding low energy structures of model proteins (Beutler (1996) ProteinSci 5(10):2037-43), and efficient algorithms for protein sequence designand the analysis of certain evolutionary fitness landscapes (Kleinberg(1999) J Comput Biol 6(3-4):387-404). In addition, other evolutionaryprotein design programs can be used, such as evolutionary algorithms incomputer-aided molecular design (Clark and Westhead (1996) J ComputAided Mol Des 10(4):337-58).

Genetic algorithms can be used to solve problems in protein threading,both in this context and generally in relation to the present invention(see also, Yadgari et al. (1998) Ismb 6:193-202). In protein threadingapproaches, a sequence is aligned and identified to the fold with whichit is most compatible (this process is often referred to in theliterature as protein “threading”). Genetic algorithms can be used tosolve such problems, which are not expected to have simple polynomialsolutions. See also, Yadgari et al, id.

4. Use protein design algorithms to assess the ability of each in silicorecombinant to fold into structures resembling the template structures.

5. Set a filtering criteria (e.g., stability, structural similarity ofone or more region of the putative cross-over construct to one or moreparental, desired regions of hydrophobicity or hydrophilicity, theability of selected regions to form particular secondary, tertiary oreven quaternary structures, or any other useful criteria) by whichrecombinants are selected or rejected depending upon their ability tofit into one or more of the template structures.

6. Identify crossover points allowed by the selected filtering criteria.

7. Design bridging oligonucleotides homologous to each position in eachpair of parents where a recombination point is allowed. Oligonucleotidedesign is optionally further refined, for example, to ensure that adisallowed crossover is not facilitated, or that all crossover oligoshave identical melting temperatures to each parent (and to all othercrossover oligos generated). For example, desirable primers optionallyincorporate any of several useful properties. These include, inter alia,that the hybridization of the primers to their complementary sequencesis uniform: that individual primers hybridize only to theircomplementary regions in the system, and do not significantly crosshybridize with primers complementary to other parental sequence regions;that if there are selected regions associated with the primers that arenot complementary to a target (e.g., cloning sites, secondary PCR primerbinding sites, etc.) that the selected regions do not hybridize to acorresponding probe set, etc. One available computer program for primerselection is the MacVector™ program from Kodak. An alternate program isthe MFOLD program (Genetics Computer Group, Madison Wis.) which predictssecondary structure of, e.g., single-stranded nucleic acids. In additionto programs for primer selection, one of skill can easily design simpleprograms for any or all of the preferred primer design steps.

8. Synthesize crossover oligonucleotides.

9. Synthesize, clone or otherwise obtain parental genes, or subsequencesthereof.

10. Fragment parental genes or subsequences (either by actual cleavageor by synthetic generation of parental gene fragments, or both).

11. Assemble mixtures of parental gene fragments and crossoveroligonucleotides.

12. Express and assay recombinants.

13. Repeat as desired to obtain an activity (new or improved) ofinterest. Note that in subsequent rounds, sequence information fromselected recombinants can be used to provide information for bridgingoligonucleotide design for subsequent steps. In addition, any selectedrecombinant can be used in any diversity generation reaction accordingto any other protocol herein (e.g., shuffling, mutagenesis, or any insilico procedure herein).

The types of enzymatic activity that arise as a result of this overallprocess depend upon the recombinants chosen and the template structurescreened against. For example, there is putative structural homologybetween Bacillus megaterium P450BM-3 and murine nitric oxide synthaseprotein (see Degtyarenko, K. N. & Archakov, A. I. (1993) FEBS 332,1-8.). Novel P450s can be computationally constructed by making allpossible recombinants between the nitric oxide synthase N-terminus andthe entire P450, following comparison with a P450 template structure toeliminate undesired sequences. Libraries of synthesized variants areconstructed that can be tested for alterations in various P450properties, e.g., substrate range, acceptable electron donors, etc.Alternatively, in this example, novel nitric oxide synthases arecomputationally constructed by making all possible recombinants betweenthe N-terminal domain of the P450 and the entire nitric oxide synthaseand comparing putative recombinants resulting from the crossovers with anitric oxide template structure for one or more property (energyminimization, stability, etc., as noted above). Desirable sequences areconstructed and functionally screened. For example, in this particularexample, a functional screen on these enzymes can screen, e.g., for adifferent property rate of electron transfer and/or nitric oxidesynthesis.

Variations on Methods for Computational Generation of Variants

A number of variations on the basic methods noted above can beperformed, including the following specifically enumerated variations.

1. In step 2 above, other ways of computationally generating new proteinsequences can be used. For example incremental truncation for thecreation of hybrid enzymes (ITCHY), circular permutations (with orwithout additional protein sequence insertions), sequence duplications,complementation, and many other techniques are applicable. Any approachthat takes naturally occurring or artificial sequences and recombinesthem in silico can be used.

For example, Ostermeier et al. “A combinatorial approach to hybridenzymes independent of DNA homology” (1999) Nat Biotechnol 17(12):1205-9describe incremental truncation for the creation of hybrid enzymes(ITCHY), that creates combinatorial fusion libraries between genes in amanner that is independent of DNA homology. The use of ITCHY and DNAshuffling to create interspecies or intraspecies fusion librariesbetween fragments of genes with little or no homology can identify amore diverse set of active fusion points including those in regions ofnonhomology and those with crossover points that diverge from a sequencealignment.

Similarly, a variety of references describe circular permutations (withor without additional protein sequence insertions). These include, e.g.,Baird et al. (1999) “Circular permutation and receptor insertion withingreen fluorescent proteins” Proc Natl Acad Sci USA 96(20):11241-6;Topell (1999) “Circularly permuted variants of the green fluorescentprotein” FEBS Lett 457(2):283-9; Hennecke et al. (1999) “Random circularpermutation of DsbA reveals segments that are essential for proteinfolding and stability” J Mol Biol 286(4):1197-215; Hennecke andGlockshuber (1998) “Conversion of a catalytic into a structuraldisulfide bond by circular permutation” Biochemistry 37(50):17590-7;Luger (1989) “Correct folding of circularly permuted variants of a betaalpha barrel enzyme in vivo” Science 243(4888):206-10; and Zhang andSchachman (1996) “In vivo formation of allosteric aspartatetranscarbamoylase containing circularly permuted catalytic polypeptidechains: implications for protein folding and assembly” Protein Sci5(7):1290-300. In brief, genes such as GFP, disulfide oxidoreductaseDsbA, aspartate transcarbamoylase (ATCase) and others have been shown tobe active even when extensively rearranged. For example, severalrearrangements of GFPs, in which the amino and carboxyl portions areinterchanged and rejoined with a short spacer connecting the originaltermini, still become fluorescent. These circular permutations havealtered pKa values and orientations of the chromophore with respect to afusion partner. Furthermore, certain locations within GFP tolerateinsertion of entire proteins, and conformational changes in the insertcan have profound effects on the fluorescence. For example, insertionsof calmodulin or a zinc finger domain in place of Tyr-145 of a yellowmutant (enhanced yellow fluorescent protein) of GFP result in indicatorproteins whose fluorescence can be enhanced upon metal binding. Thecalmodulin graft into enhanced yellow fluorescent protein can monitorcytosolic Ca(2+) in single mammalian cells. The tolerance of proteinsfor circular permutations and insertions shows that the folding processis robust and offers a general strategy for creating new diversifiedsequences, including completely non-homologous sequences which arejoined by bridging oligonucleotides according to the present invention.

In one aspect, active sequences are produced by complementation. Forexample, Yang and Schachman (1993) “In vivo formation of activeaspartate transcarbamoylase from complementing fragments of thecatalytic polypeptide chains” Protein Sci 2(6):1013-23 and, e.g., Yangand Schachman (1996) “A bifunctional fusion protein containing themaltose-binding polypeptide and the catalytic chain of aspartatetranscarbamoylase: assembly, oligomers, and domains” Biophys Chem59(3):289-97 describe formation of an active stable enzyme in vivo, evenwith fragmented catalytic chains. Thus, as applied to the presentinvention, domains or other subsequences can be separately designed andsynthesized and tested for complementary effects with one another.Because the domains/sequences are on separate chains, complementationcan be used to assess combinatorial effects of the separate domains.This approach can increase the number of sequence combinations that areassessed for activity, without increasing the number of sequences thatare actually synthesized.

2. Incomplete protein fragments or putative protein domains (e.g. fromESTs) can also be included in any recombination reaction. Domains can beapproximately identified, or even arbitrarily designated, e.g., asdescribed supra. Additional details regarding protein domaindesignation/identification are found, e.g., in Crameri et al.“OLIGONUCLEOTIDE MEDIATED NUCLEIC ACID RECOMBINATION” Filed Jan. 18,2000; U.S. Ser. No.: PCT/US00/01203.

For example, in the p450 example noted, there are at least 6 differentforms of P450 known (see Degtyarenko and Archakov (1993) FEBS 332, 1-8;see also, Ortiz de Montellano (ed.) (1995) Cytochrome P450 StructureMechanism and Biochemistry. Second Edition Plenum Press (New York andLondon) and the references cited therein). For example, as shown byDegtyarenko and Archakov, the molecular evolution of P450s, in contrastto that of many protein families, does not follow phylogeny. During theevolution of P450s, gene duplications and gene fusions, horizontal genetransfer and intron loss events have occurred. ‘Weak’ and ‘strong’hierarchies in the clustering of P450 sequences can be shown.

The six different forms of p450s include prokaryotic and eukaryotic 3part systems with separate FAD-containing, Fe—S and P450(heme-containing) subunits; prokaryotic and eukaryotic 2 part systemswith a combined FAD- and FMN-containing subunit and a P450(heme-containing) subunit; at least one prokaryotic one-component system(Bacillus megaterium P450BM-3) with one polypeptide containing FAD- FMN-and heme-group, and at least one eukaryotic one-component system (murinenitric oxide synthase) with one polypeptide containing FAD- FMN- andheme-group.

Thus, the combined FAD- and FMN-containing subunit from eitherprokaryotic or eukaryotic 2-component systems are optionallycomputationally recombined with the FAD- FMN-containing domain of eitherof the one-component systems, and then tested against a one-componenttemplate structure. Likewise, the P450 subunits from either the 2 or 3component systems can be computationally recombined with theheme-containing domain of the one-component system and then testedagainst a one-component template structure. The converse can also beperformed, i.e., a domain from the one-component system cancomputationally recombined with a subunit from the multi-componentsystems, and screened in silico for one or more computed property, e.g.against a structure of one of the subunits as a template.

3. Potential sequences for recombination can also be identified bycomputational methods other than by direct homology or structuralinformation, e.g. by the PRINTS system (Attwood T K Beck M E, Bleasby AJ, Degtyarenko K, Parry Smith D J Nucleic Acids Res 1996 Jan. 1; 24(1):182-8), Shotgun (Pegg and Babbitt (1999) Bioinformatics 15(9):729-40)independently evolving sequence modules (may correspond to folding unitsas in MasterCatalog (eragen.com, on the world wide web) and othersequence comparison methods.

For example, Pegg and Babbitt (id.) provide an example of using theShotgun program to identify both new superfamily members and toreconstruct known enzyme superfamilies, using BLAST database searches.An analysis of the false-positive rates generated in the analysis andother control experiments show that high Shotgun scores indicateevolutionary relationships. Shotgun is also a useful tool foridentifying subgroup relationships within superfamilies and for testinghypotheses about related protein families.

Similarly, PRINTS (e.g., Atwood et al., above) is a compendium ofprotein motif fingerprints derived from the OWL composite sequencedatabase. Fingerprints are groups of motifs within sequence alignmentswhose conserved nature allows them to be used as signatures of familymembership. Fingerprints can provide improved diagnostic reliabilityover single motif methods by virtue of the mutual context provided bymotif neighbors. The database is now accessible via the UCLBioinformatics Server on biochem.ucl.ac.uk./bsm/dbbrowser/ (on the worldwide web). Atwood et al. describe the database, its compilation andinterrogation software, and its Web interface. See also, Attwood et al.(1997) “Novel developments with the PRINTS protein fingerprint database”Nucleic Acids Res 25(1):212-7.

4. In addition to calculating all possible single crossovers for allpairwise combinations of parental genes, it is also possible tocalculate multiple crossovers and compare those with the templatestructures. In this case it is possible to identify crossover oligosthat should be used in combination to provide any computationallyselected criteria.

Similarly, it is also possible to take all possible pairwiserecombinants which are generated and then run all possible pairwisecombinations of those recombinants with each other, or with otherparents. This is useful if the program has an output feature where thenumber of bridging oligos for the different possible combinations ofmultiple parents are described. For example in a family of proteins A-F,A, B and D may have many productive crossovers with each other, A C, Eand F may have many productive crossovers with each other, but there maybe few between (B or D) and (C, E and F). This information is helpful indefining which combinations of parents are actually mixed when buildingthe physical libraries.

5. Crossover points can also be “calculated” by simply comparing thestructures (either from crystals, nmr, dynamic simulations, or any otheravailable method) of proteins corresponding to nucleic acids to berecombined. All possible pairwise combinations of structures can beoverlaid. Amino acids can be identified as possible crossover pointswhen they overlap with each other on the parental structures (this candefine closeness of fit; for example, the alpha carbons can be selectedto be within 1 angstrom), or when they and their nearest neighboursoverlap within similar distance criteria. Bridging oligos can be builtfor each crossover point. Thus, in this embodiment, in silico selectionof recombinants and the step of cross-over selection in parentalsequences are combined into a single simultaneous step.

An advantage to this approach is that structural analysis can bebypassed for resulting chimeras. This is because the chimeras havealready been “pre-selected” for structural features that correspond tothe parental sequences.

Variations on Structural Calculation Methods

Variations on the basic approach noted above can also be performed byvarying calculation of structural information. Variations include thefollowing.

1. The entire process can be conducted at the level of sequence domains,e.g., by identifying domains in a structure and then identifyinghomologous domains for each target domain. Calculations can be made forthe ability of in silico recombinants to form a complete folded protein,or for any given domain to form a structural domain.

Using the one-component Bacillus megaterium P450BM-3 example, the FAD-and FMN-containing domain and the P450 (heme containing) domain can betreated as structurally separable. Thus the FAD- and FMN-containingdomain can be computationally recombined with the FAD- andFMN-containing domain of murine nitric oxide synthase, the FAD- andFMN-containing subunit of 2-component systems, and the FAD-containingsubunit of 3-component systems. These can be fit either to a completeP450BM-3 template structure, or to a template structure that onlyincludes the FAD- and FMN-containing domain and is not constrained bythe presence of the heme-containing domain.

In this way all possible computational recombinants can be computed foreach domain of a protein, and then these domains can be computationallycombined, followed by synthesis of appropriate cross-overoligonucleotides. This reduces the complexity of the overallcomputational problem of recombining low similarity sequences.

2. Other known structures (e.g., for proteins other than the parentalstructures) can also be used as template structures. In addition,calculated structures can also be used. This can include, e.g., any ofthe sequences or sequence intermediates described herein, includingevolutionary intermediates, diplomat sequences, and the like.

Formats for Making and Using Bridging Oligonucleotides to Facilitate LowHomology Recombination

As noted, this invention provides, inter alia, a process by whichvariants produced computationally can be synthesized physically, withoutthe need for massive parallel synthesis of each individual calculatedgene. Parental genes can be cloned or synthesized and then fragmented(e.g., by the uracil cleavage methods noted supra, e.g., to allow formulti-library formats as noted below), or fragments of the parents cansimply be synthesized. The fragments are mixed with the relevantcrossover oligos, assembled, expressed and assayed. Instead ofindividually synthesizing each in silico identified gene, oneoligonucleotide is sufficient for each crossover oligonucleotide. Manydifferent progeny can then be synthesized from gene fragments andcrossover oligonucleotides simply by assembling different combinationsof parents and oligonucleotides. The actual synthesis is performedeither as each individual variant synthesized independently, or inincreasing pool sizes up to a single library of all variants, e.g., byusing, a 96-well parallel format (or other common screening format).

For example, the following steps can be used in one basic format of thismethod.

1. Each variant is synthesized, e.g., in a single microtitre well, e.g.,by including two parents and one bridging oligonucleotide.

2. Selected (e.g., calculated to be useful, e.g., according to anyselected set of criteria) crossover oligonucleotides are synthesized,representing different pairs of parents. For example, for 5 parents,there are 10 different pairwise combinations of crossoveroligonucleotides.

3. Libraries containing all parents but a different oligonucleotide ateach position are synthesized.

4. Oligonucleotides are included in the same reaction with all relevantparents to generate all selected crossovers.

Combinations of the above steps can be performed, separately orindividually and in different orders.

In the case of permutated or truncated parental sequences, it is usefulto synthesize, clone or otherwise construct the truncated or permutatedparental sequences, as well as the bridging oligonucleotides.

Iterative Variations

The method can also incorporate a variety of variations to produce aniterative process. The following are example variations.

1. Improved variants from the first round of recombination can beshuffled by any of the processes noted herein, including use or bridgingor cross-over oligonucleotides.

2. The computation itself can be iteratively improved. For example,first round improved variants can be computationally fitted to theparental structures used, and the improved structures can then becalculated, e.g., by energy minimization. New structures of the improvedvariants are, e.g., used as template structures for second roundcalculated recombinants. In this way it is also possible to gainstructural understanding of functional changes which are obtained.Neural net and/or other statistical approaches can be used to furtherrefine second round recombinants.

3. This approach is productively combined with the formats describedabove that do not produce single pooled libraries. For example, taking,e.g., 5 genes, 10 possible pairwise libraries are synthesized. Eachlibrary is separately screened for hits. Picking one or a few hits fromeach library identifies tens to hundreds of different recombinants(because they come from different libraries). Comparisons of thecalculated structures of these recombinants can be used to identifyregions of structural constraint and regions where there is moreflexibility. The second round of in silico recombinants can then becompared with all of these calculated structures as a second generationof templates. This identifies parts of the structure that are relevantfor function, and ensures that recombinants are rejected in silico wherethey deviate from template structural features that are relevant for thefunction under consideration. Alternatively, a “consensus” templatestructure can be built by giving different importance weightings todifferent parts of the protein, and a single structure used.

Non-Oligonucleotide Dependant in Silico Shuffling

As discussed herein, many of the methods of the invention involvegenerating diversity in sequence strings in silico, followed byoligonucleotide gene recombination/synthesis methods. However,non-oligonucleotide based recombination methods are also appropriate.For example, instead of generating oligonucleotides, entire genes can bemade which correspond to any diversity created in silico, without theuse of oligonucleotide intermediates. This is particularly feasible whengenes are sufficiently short that direct synthesis is possible.

In addition, it is possible to generate peptide sequences directly fromdiverse character string populations, rather than going througholigonucleotide intermediates. For example, solid phase polypeptidesynthesis can be performed. For example, solid phase peptide arrays canbe constructed by standard solid phase peptide synthesis methods, withthe members of the arrays being selected to correspond to the in silicogenerated sequence strings.

In this regard, solid phase synthesis of biological polymers, includingpeptides has been performed at least since the early “Merrifield” solidphase peptide synthesis methods, described, e.g. in Merrifleld (1963) J.Am. Chem. Soc. 85:2149-2154 (1963). Solid-phase synthesis techniques areavailable for the synthesis of several peptide sequences on, forexample, a number of “pins.” See e.g., Geysen et al. (1987) J. Immun.Meth. 102:259-274, incorporated herein by reference for all purposes.Other solid-phase techniques involve, for example, synthesis of variouspeptide sequences on different cellulose disks supported in a column.See. Frank and Doring (1988) Tetrahedron 44:6031-6040. Still othersolid-phase techniques are described in U.S. Pat. No. 4,728,502 issuedto Hamill and WO 90/00626. Methods of forming large arrays of peptidesare also available. For example, Pirrung et al., U.S. Pat. No. 5,143,854and Fodor et al., PCT Publication No. WO 92/10092, disclose methods offorming arrays of peptides and other polymer sequences using, forexample, light-directed synthesis techniques. See also, Stewart andYoung, Solid Phase Peptide Synthesis, 2d. ed., Pierce Chemical Co.(1984); Atherton et al. (1989) Solid Phase Peptide Synthesis, IRL Press,Greene, et al. (1991) Protective Groups In Organic Chemistry, 2nd Ed.,John Wiley & Sons, New York, N.Y. and Bodanzszyky (1993) Principles ofPeptide Synthesis second edition Springer Verlag, Inc. NY. Other usefulinformation regarding proteins is found in R. Scopes, ProteinPurification, Springer-Verlag, N.Y. (1982); Deutscher, Methods inEnzymology Vol. 182: Guide to Protein Purification, Academic Press, Inc.N.Y. (1990); Sandana (1997) Bioseparation of Proteins, Academic Press,Inc.; Bollag et al. (1996) Protein Methods, 2^(nd) Edition Wiley-Liss,NY; Walker (1996) The Protein Protocols Handbook Humana Press, N.J.),Harris and Angal (1990) Protein Purification Applications: A PracticalApproach IRL Press at Oxford, Oxford, England; Harris and Angal ProteinPurification Methods: A Practical Approach IRL Press at Oxford, Oxford,England; Scopes (1993) Protein Purification: Principles and Practice3^(rd) Edition Springer Verlag, NY; Janson and Ryden (1998) ProteinPurification: Principles, High Resolution Methods and Applications,Second Edition Wiley-VCH, NY; and Walker (1998) Protein Protocols onCD-ROM Humana Press, NJ; and the references cited therein.

In addition to proteins and nucleic acids, it should be appreciated thatcharacter string diversity generated in silico can be corresponded toother biopolymers. For example, the character strings can becorresponded to peptide nucleic acids (PNAs) which can be synthesizedaccording to available techniques and screened for activity in anyappropriate assay. See, e.g., Peter E. Nielsen and Michael Egholm (eds)(1999) Peptide Nucleic Acids: Protocols and Applications ISBN1-898486-16-6 Horizon Scientific Press, Wymondham, Norfolk, U.K for anintroduction to PNA synthesis and activity screening.

Assays—Physical Selection.

Directed Evolution by GAGGS, as in DNA shuffling, or classical strainimprovement, or any functional genomics technology, can use any physicalassays known in the art for detecting polynucleotides encoding desiredphenotypes.

Synthetic genes are amenable to conventional cloning and expressionapproaches; thus, properties of the genes and proteins they encode canreadily be examined after their expression in a host cell. Syntheticgenes can also be used to generate polypeptide products by in-vitro(cell-free) transcription and translation. Polynucleotides andpolypeptides can thus be examined for their ability to bind a variety ofpredetermined ligands, small molecules and ions, or polymeric andheteropolymeric substances, including other proteins and polypeptideepitopes, as well as microbial cell walls, viral particles, surfaces andmembranes.

For example, many physical methods can be used for detectingpolynucleotides encoding phenotypes associated with catalysis ofchemical reactions by either polynucleotides directly, or by encodedpolypeptides. Solely for the purpose of illustration, and depending onspecifics of particular pre-determined chemical reactions of interest,these methods may include a multitude of techniques well known in theart which account for a physical difference between substrate(s) andproduct(s), or for changes in the reaction media associated withchemical reaction (e.g. changes in electromagnetic emissions,adsorption, dissipation, and fluorescence, whether UV, visible orinfrared (heat). These methods also can be selected from any combinationof the following: mass-spectrometry; nuclear magnetic resonance;isotopically labeled materials, partitioning and spectral methodsaccounting for isotope distribution or labeled product formation;spectral and chemical methods to detect accompanying changes in ion orelemental compositions of reaction product(s) (including changes in pH,inorganic and organic ions and the like). Other methods of physicalassays, suitable for use in GAGGS, can be based on the use of biosensorsspecific for reaction product(s), including those comprising antibodieswith reporter properties, or those based on in vivo affinity recognitioncoupled with expression and activity of a reporter gene. Enzyme-coupledassays for reaction product detection and cell life-death-growthselections in vivo can also be used where appropriate. Regardless of thespecific nature of the physical assays, they all are used to select adesired property, or combination of desired properties, encoded by theGAGGS-generated polynucleotides. Polynucleotides found to have desiredproperties are thus selected from the library.

The methods of the invention optionally include selection and/orscreening steps to select nucleic acids having desirablecharacteristics. The relevant assay used for the selection will dependon the application. Many assays for proteins, receptors, ligands and thelike are known. Formats include binding to immobilized components, cellor organismal viability, production of reporter compositions, and thelike.

In high throughput assays, it is possible to screen up to severalthousand different shuffled variants in a single day. For example, eachwell of a microtiter plate can be used to run a separate assay, or, ifconcentration or incubation time effects are to be observed, every 5-10wells can test a single variant (e.g., at different concentrations).Thus, a single standard microtiter plate can assay about 100 (e.g., 96)reactions. If 1536 well plates are used, then a single plate can easilyassay from about 100-about 1500 different reactions. It is possible toassay several different plates per day; assay screens for up to about6,000-20,000 different assays (i.e., involving different nucleic acids,encoded proteins, concentrations, etc.) is possible using the integratedsystems of the invention. More recently, microfluidic approaches toreagent manipulation have been developed, e.g., by Caliper Technologies(Mountain View, Calif.) which can provide very high throughputmicrofluidic assay methods.

In one aspect, cells, viral plaques, spores or the like, comprisingGAGGS shuffled nucleic acids, are separated on solid media to produceindividual colonies (or plaques). Using an automated colony picker(e.g., the Q-bot, Genetix, U.K.), colonies or plaques are identified,picked, and up to 10,000 different mutants inoculated into 96 wellmicrotiter dishes containing two 3 mm glass balls/well. The Q-bot doesnot pick an entire colony but rather insects a pin through the center ofthe colony and exits with a small sampling of cells, (or mycelia) andspores (or viruses in plaque applications). The time the pin is in thecolony, the number of dips to inoculate the culture medium, and the timethe pin is in that medium each effect inoculum size, and each parametercan be controlled and optimized.

The uniform process of automated colony) picking such as the Q-botdecreases human handling error and increases the rate of establishingcultures (roughly 10,000/4 hours). These cultures are optionally shakenin at temperature and humidity controlled incubator. Optional glassballs in the microtiter plates act to promote uniform aeration of cellsand the dispersal of cellular (e.g., mycelial) fragments similar to theblades of a fermenter. Clones from cultures of interest can be isolatedby limiting dilution. As also described supra, plaques or cellsconstituting libraries can also be screened directly for the productionof proteins, either by detecting hybridization, protein activity,protein binding to antibodies, or the like. To increase the chances ofidentifying a pool of sufficient size, a prescreen that increases thenumber of mutants processed by 10-fold can be used. The goal of theprimary screen is to quickly identify mutants having equal or betterproduct titers than the parent strain(s) and to move only these mutantsforward to liquid cell culture for subsequent analysis.

One approach to screening diverse libraries is to use a massivelyparallel solid-phase procedure to screen cells expressing shufflednucleic acids, e.g., which encode enzymes for enhanced activity.Massively parallel solid-phase screening apparatus using absorption,fluorescence, or FRET are available. See, e.g., U.S. Pat. No. 5,914,245to Bylina, et al. (1999); see also, kairos-scientific.com/ (on the worldwide web); Youvan et al. (1999) “Fluorescence ImagingMicro-Spectrophotometer (FIMS)” Biotechnology et alia<et-al.com (on theworld wide web)> 1:1-16; Yang et al. (1998) “High Resolution ImagingMicroscope (HIRIM)” Biotechnology et alia, <et-al.com (on the world wideweb)> 4:1-20; and Youvan et al. (1999) “Calibration of FluorescenceResonance Energy Transfer in Microscopy Using Genetically Engineered GFPDerivatives on Nickel Chelating Beads” posted at kairos-scientific.com(on the world wide web). Following screening by these techniques,sequences of interest are typically isolated, optionally sequenced andthe sequences used as set forth herein to design new sequences for insilico or other shuffling methods.

Similarly, a number of well known robotic systems have also beendeveloped for solution phase chemistries useful in assay systems. Thesesystems include automated workstations like the automated synthesisapparatus developed by Takeda Chemical Industries, LTD. (Osaka, Japan)and many robotic systems utilizing robotic arms (Zymate II, ZymarkCorporation, Hopkinton, Mass.; Orca, Beckman Coulter, Inc. (Fullerton,Calif.)) which mimic the manual synthetic operations performed by ascientist. Any of the above devices are suitable for use with thepresent invention, e.g., for high-throughput screening of moleculesencoded by codon-altered nucleic acids. The nature and implementation ofmodifications to these devices (if any) so that they can operate asdiscussed herein will be apparent to persons skilled in the relevantart.

High throughput screening systems are commercially available (see, e.g.,Zymark Corp., Hopkinton, Mass.; Air Technical Industries, Mentor, Ohio;Beckman Instruments, Inc. Fullerton, Calif.; Precision Systems, Inc.,Natick, Mass., etc.). These systems typically automate entire proceduresincluding all sample and reagent pipetting, liquid dispensing, timedincubations, and final readings of the microplate in detector(s)appropriate for the assay. These configurable systems provide highthroughput and rapid start up as well as a high degree of flexibilityand customization.

The manufacturers of such systems provide detailed protocols the varioushigh throughput. Thus, for example, Zymark Corp. provides technicalbulletins describing screening systems for detecting the modulation ofgene transcription, ligand binding, and the like.

A variety of commercially available peripheral equipment and software isavailable for digitizing, storing and analyzing a digitized video ordigitized optical or other assay images, e.g., using PC (Intel x86 orpentium chip-compatible DOS™, OS2™ WINDOWS™, WINDOWS NT™ or WINDOWS95™based machines), MACINTOSH™, or UNIX based (e.g., SUN™ work station)computers.

Integrated systems for analysis typically include a digital computerwith GO software for GAGGS, and, optionally, high-throughput liquidcontrol software, image analysis software, data interpretation software,a robotic liquid control armature for transferring solutions from asource to a destination operably linked to the digital computer, aninput device (e.g., a computer keyboard) for entering data to thedigital computer to control GAGGS operations or high throughput liquidtransfer by the robotic liquid control armature and, optionally, animage scanner for digitizing label signals from labeled assaycomponents. The image scanner can interface with image analysis softwareto provide a measurement of probe label intensity. Typically, the probelabel intensity measurement is interpreted by the data interpretationsoftware to show whether the labeled probe hybridizes to the DNA on thesolid support.

Current art computational hardware resources are fully adequate forpractical use in GAGGS (any mid-range priced Unix system (e.g., for SunMicrosystems) or even higher end Macintosh or PCs will suffice). Currentart in software technology is adequate (i.e., there are a multitude ofmature programming languages and source code suppliers) for design of anupgradeable open-architecture object-oriented genetic algorithm package,specialized for GAGGS users with a biological background.

A Digital Apparatus for GOs

Various methods and genetic algorithms (GOs) can be used to performdesirable functions as noted herein. In addition, digital or analogsystems such as digital or analog computer systems can control a varietyof other functions such as the display and/or control of output files.

For example, standard desktop applications such as word processingsoftware (e.g., Microsoft Word™ or Corel WordPerfect™) and databasesoftware (e.g., spreadsheet software such as Microsoft Excel™, CorelQuattro Pro™, or database programs such as Microsoft Access™ orParadox™) can be adapted to the present invention by inputting one ormore character string into the software which is loaded into the memoryof a digital system, and performing a GO as noted herein on thecharacter string. For example, systems can include the foregoingsoftware having the appropriate character string information, e.g., usedin conjunction with a user interface (e.g., a GUI in a standardoperating system such as a Windows, Macintosh or LINUX system) tomanipulate strings of characters, with GOs being programmed into theapplications, or with the GOs being performed manually by the user (orboth). As noted, specialized alignment programs such as PILEUP and BLASTcan also be incorporated into the systems of the invention, e.g., foralignment of nucleic acids or proteins (or corresponding characterstrings) as a preparatory step to performing an additional GO on theresulting aligned sequences. Software for performing PCA can also beincluded in the digital system.

Systems for GO manipulation typically include, e.g., a digital computerwith GO software for aligning and manipulating sequences according tothe GOs noted herein, or for performing PCA, or the like, as well asdata sets entered into the software system comprising sequences to bemanipulated. The computer can be, e.g., a PC (Intel x86 or Pentiumchip-compatible DOS,™ OS2,™ WINDOWS,™ WINDOWS NT,™ WINDOWS95,™WINDOWS98,™ LINUX, Apple-compatible, MACINTOSH™ compatible, Power PCcompatible, or a UNIX compatible (e.g., SUN™ work station) machine) orother commercially common computer which is known to one of skill.Software for aligning or otherwise manipulating sequences can beconstructed by one of skill using a standard programming language suchas Visualbasic, Fortran, Basic, Java, or the like, according to themethods herein.

Any controller or computer optionally includes a monitor which caninclude, e.g., a cathode ray tube (“CRT”) display, a flat panel display(e.g., active matrix liquid crystal display, liquid crystal display), orothers. Computer circuitry is often placed in a box which includesnumerous integrated circuit chips, such as a microprocessor, memory,interface circuits, and others. The box also optionally includes a harddisk drive, a floppy disk drive, a high capacity removable drive such asa writeable CD-ROM, and other common peripheral elements. Inputtingdevices such as a keyboard or mouse optionally provide for input from auser and for user selection of sequences to be compared or otherwisemanipulated in the relevant computer system.

The computer typically includes appropriate software for receiving userinstructions, either in the form of user input into a set parameterfields, e.g., in a GUI, or in the form of preprogrammed instructions,e.g., preprogrammed for a variety of different specific operations. Thesoftware then converts these instructions to appropriate language forinstructing the system to carry out any desired operation. For example,in addition to performing GO manipulation of character strings, adigital system can instruct an oligonucleotide synthesizer to synthesizeoligonucleotides for gene reconstruction, or even to orderoligonucleotides from commercial sources (e.g., by printing appropriateorder forms or by linking to an order form on the internet).

The digital system can also include output elements for controllingnucleic acid synthesis (e.g., based upon a sequence or an alignment of asequences herein), i.e., an integrated system of the inventionoptionally includes an oligonucleotide synthesizer or an oligonucleotidesynthesis controller. The system can include other operations whichoccur downstream from an alignment or other operation performed using acharacter string corresponding to a sequence herein, e.g., as notedabove with reference to assays.

In one example, GOs of the invention are embodied in a fixed media ortransmissible program component containing logic instructions and/ordata that when loaded into an appropriately configured computing devicecauses the device to perform a GO on one or more character string. FIG.13 shows example digital device 700 that should be understood to be alogical apparatus that can read instructions from media 717, networkport 719, user input keyboard 709, user input 711 or other inputtingmeans. Apparatus 700 can thereafter use those instructions to direct GOmodification of one or more character string, e.g., to construct one ormore data set (e.g. comprising a plurality of GO modified sequencescorresponding to nucleic acids or proteins). One type of logicalapparatus that can embody the invention is a computer system as incomputer system 700 comprising CPU 707, optional user input deviceskeyboard 709, and GUI pointing device 711, as well as peripheralcomponents such as disk drives 715 and monitor 705 (which displays GOmodified character strings and provides for simplified selection ofsubsets of such character strings by a user. Fixed media 717 isoptionally used to program the overall system and can include, e.g., adisk-type optical or magnetic media or other electronic memory storageelement. Communication port 719 can be used to program the system andcan represent any type of communication connection.

The invention can also be embodied within the circuitry of anapplication specific integrated circuit (ASIC) or programmable logicdevice (PLD). In such a case, the invention is embodied in a computerreadable descriptor language that can be used to create an ASIC or PLD.The invention can also be embodied within the circuitry or logicprocessors of a variety of other digital apparatus, such as PDAs, laptopcomputer systems, displays, image editing equipment, etc.

In one preferred aspect, the digital system comprises a learningcomponent where the outcomes of physical oligonucleotide assemblyschemes (compositions, abundance of products, different processes) aremonitored in conjunction with physical assays, and correlations areestablished. Successful and unsuccessful combinations are documented ina database to provide justification/preferences for user-base or digitalsystem based selection of sets of parameters for subsequent GAGGSprocesses involving the same set of parental character strings/nucleicacids/proteins (or even unrelated sequences, where the informationprovides process improvement information). The correlations are used tomodify subsequent GAGGS processes to optimize the process. This cycle ofphysical synthesis, selection and correlation is optionally repeated tooptimize the system. For example, a learning neural network can be usedto optimize outcomes.

Embodiment in a Web Site.

The methods of this invention can be implemented in a localized ordistributed computing environment. In a distributed environment, themethods may implemented on a single computer comprising multipleprocessors or on a multiplicity of computers. The computers can belinked, e.g. through a common bus, but more preferably the computer(s)are nodes on a network. The network can be a generalized or a dedicatedlocal or wide-area network and, in certain preferred embodiments, thecomputers may be components of an intra-net or an internet.

In one internet embodiment a client system typically executes a Webbrowser and is coupled to a server computer executing a Web server. TheWeb browser is typically a program such as IBM's Web Explorer, Internetexplorer, NetScape or Mosaic. The Web server is typically, but notnecessarily, a program such as IBM's HTTP Daemon or other WWW daemon(e.g., LINUX-based forms of the program). The client computer isbi-directionally coupled with the server computer over a line or via awireless system. In turn, the server computer is bi-directionallycoupled with a website (server hosting the website) providing access tosoftware implementing the methods of this invention.

A user of a client connected to the Intranet or Internet may cause theclient to request resources that are part of the web site(s) hosting theapplication(s) providing an implementation of the methods of thisinvention. Server program(s) then process the request to return thespecified resources (assuming they are currently available). A standardnaming convention has been adopted, known as a Uniform Resource Locator(“URL”). This convention encompasses several types of location names,presently including subclasses such as Hypertext Transport Protocol(“http”), File Transport Protocol (“ftp”), gopher, and Wide AreaInformation Service (“WAIS”). When a resource is downloaded, it mayinclude the URLs of additional resources. Thus, the user of the clientcan easily learn of the existence of new resources that he or she hadnot specifically requested.

The software implementing the method(s) of this invention can runlocally on the server hosting the website in a true client-serverarchitecture. Thus, the client computer posts requests to the hostserver which runs the requested process(es) locally and then downloadsthe results back to the client. Alternatively, the methods of thisinvention can be implemented in a “multi-tier” format wherein acomponent of the method(s) are performed locally by the client. This canbe implemented by software downloaded from the server on request by theclient (e.g. a Java application) or it can be implemented by software“permanently” installed on the client.

In one embodiment the application(s) implementing the methods of thisinvention are divided into frames. In this paradigm, it is helpful toview an application not so much as a collection of features orfunctionality but, instead, as a collection of discrete frames or views.A typical application, for instance, generally includes a set of menuitems, each of with invokes a particular frame—that is, a form whichmanifest certain functionality of the application. With thisperspective, an application is viewed not as a monolithic body of codebut as a collection of applets, or bundles of functionality. In thismanner from within a browser, a user would select a Web page link whichwould, in turn, invoke a particular frame of the application (i.e.,subapplication). Thus, for example, one or more frames may providefunctionality for inputting and/or encoding biological molecule(s) intoone or more character strings, while another frame provides tools forgenerating and/or increasing diversity of the encoded characterstring(s).

In particular preferred embodiments, the methods of this invention areimplemented as one or more frames providing, e.g., the followingfunctionialit(ies). Function(s) to encode two or more biologicalmolecules into character strings to provide a collection of two or moredifferent initial character strings wherein each of said biologicalmolecules comprises a selected set of subunits; functions to select atleast two substrings from the character strings; functions toconcatenate the substrings to form one or more product strings about thesame length as one or more of the initial character strings; functionsto add (place) the product strings to a collection of strings, andfunctions to implement any feature of GAGGS or any GO or GA as set forthherein.

The functions to encode two or more biological molecules can provide oneor more windows wherein the user can insert representation(s) ofbiological molecules. In addition, the encoding function also,optionally, provides access to private and/or public databasesaccessible through a local network and/or the intranet whereby one ormore sequences contained in the databases can be input into the methodsof this invention. Thus, for example, in one embodiment, where the enduser inputs a nucleic acid sequenced into the encoding function, theuser can, optionally, have the ability to request a search of GenBankand input one or more of the sequences returned by such a search intothe encoding and/or diversity generating function.

Methods of implementing Intranet and/or Intranet embodiments ofcomputational and/or data access processes are well known to those ofskill in the art and are documented in great detail (see, e.g., Cluer etal. (1992) A General Framework for the Optimization of Object-OrientedQueries, Proc SIGMOD International Conference on Management of Data, SanDiego, Calif., Jun. 2-5, 1992, SIGMOD Record, vol. 21, Issue 2, June,1992; Stonebraker, M., Editor; ACM Press, pp. 383-392; ISO-ANSI, WorkingDraft, “Information Technology-Database Language SQL”, Jim Melton,Editor, International Organization for Standardization and AmericanNational Standards Institute, Jul. 1992; Microsoft Corporation, “ODBC2.0 Programmer's Reference and SDK Guide. The Microsoft Open DatabaseStandard for Microsoft Windows.™, and Windows NT.™., Microsoft OpenDatabase Connectivity™ Software Development Kit”, 1992, 1993, 1994Microsoft Press, pp. 3-30 and 41-56; ISO Working Draft, “Databaselanguage SQL-Part 2: Foundation (SQL/Foundation)”, CD9075-2:199.chi.SQL.Sep. 11, 1997, and the like). Additional relevant details regarding webbased applications are found in “METHODS OF POPULATING DATA STRUCTURESFOR USE IN EVOLUTIONARY SIMULATIONS” by Selifonov and Stemmer,PCT/US00/01138.

EXAMPLES

The following examples are intended to further illustrate the presentinvention and should not be considered to be limiting. One of skill willimmediately recognize a variety of parameters which can be changed toachieve essentially similar results.

Example 1 Decision Tree for Example GAGGS Process

A set of flow schematics which provide a general representation of anexemplary process of Directed Evolution (DE) by GAGGS are enclosed(FIGS. 1-4). FIG. 1 provides an example decision making process from anidea of a desired property to selection of a genetic algorithm. FIG. 2provides a directed evolution decision tree from selection of thegenetic algorithm to a refined library of parental character strings.FIG. 3 provides example processing steps from the refined parentallibrary to a raw derivative library of character strings. FIG. 4processes the raw character strings to strings with a desired property.

Generally the charts are schematics of arrangements for components, andof process decision tree structures. It is apparent that manymodifications of this particular arrangement for DEGAGGS, e.g., as setforth herein, can be developed and practiced. Certain quality controlmodules and links, as well as most of the generic artificial neuralnetwork learning components are omitted for clarity, but will beapparent to one of skill. The charts are in a continuous arrangement,each connectable head-to tail. Additional material and implementation ofindividual GO modules, and many arrangements of GOs in working sequencesand trees, as used in GAGGS, are available in various software packages.Suitable references describing exemplar existing software are found,e.g., at aic.nrl.navy.mil/galist/ (on the world wide web) and atcs.purdue.edu/coast/archive/clife/FAQ/www/Q20_(—)2.htm (on the worldwide web). It will be apparent that many of the decision stepsrepresented in FIGS. 1-4 are performed most easily with the assistanceof a computer, using one or more software program to facilitateselection/ decision processes.

Example 2 Modeling Cost Estimates

Use of degenerate synthetic oligos with very limited degree/low level ofpositional degeneracy (under 0.01-5% per position) can offer a verysubstantial cost saving in building those libraries which incorporatesubstantial mutagenicity. For PCR assembly gene synthesis, however,representation of all of the crossover events between parental entriesuses synthesis of two dedicated oligos per simulated crossover event.

However, as will be apparent from the examples below and from thecombinatorial nature of nucleic acid evolution algorithms, even buildingvery large (10⁹-10¹⁰) gene libraries for physical screening uses lessthin 10³ individual 40-mer oligos for evolution of a family of typicalgenes of ˜1.6 kb size.

Several typical examples below provide examples of costs of genesynthesis components in GAGGS, where the cost calculation is basedarbitrarily at $0.7 per base (for a 40-50 nmol quantity, which isadequate for gene reassembly procedures) for exemplary purposes. Largervolume demand in oligo synthesis service leads to substantially lowerunit cost (e.g., to a decrease of as much as 10 fold) and the generalcosts of oligo synthesis are in decline. Oligo synthesis is aninheritantly parallel and routine process easily amenable to automationand thus to increases in throughput. Currently, non-chip paralleldevices for oligo synthesis provide an effective capacity to completesimultaneous (single-load) synthesis of 196 (2×96) individual 60-meroligos in less than 5 hours, with the cost of hardware under $100 K, andthe cost of reagents under $0.07 per base. Therefore, with anunderstanding of these costs, the cost estimates made in the examplesbelow can be reduced by at least 8 fold.

Example 3 GAGGS of a Single Parent Low Mutagenicity Library

This example describes GAGGS of a single parent low mutagenicity libraryderived from an average gene (˜1.6 kb), given the sequence informationof a single 1.6 kb gene (encoding 500 aa+“convenience” start/endoligos). The goal is to build a library of gene variants with allpossible single amino acid changes, one aa change per each gene copy inthe library.

Relevant parameters include the number of oligos and cost to build 1parental 1.6 kb gene, e.g., from 40 mer oligos, with complete 20+20 baseoverlaps e.g., by non-error prone assembly PCR, the number of allpossible single aa replacement mutations, the number of distinctnon-degenerate 40-mer oligos used to “build-in” all possible single aamutations, the minimal number of all distinct fixed-positionsingle-codon-degenerate oligos used to incorporate all possible singleaa mutations, but not terminations, and the minimal number of alldistinct fixed-position single-codon-fully degenerate oligos used toincorporate all possible single aa mutations.

For a 1.6 kb gene, 1 ∞ 1,600:40 ∞ 2=80 oligos; $0.7 ∞ 40 ∞ 80=$2,240.N=500 ∞ 19=9,500 9.500×2=19,000; $532,000 @ $0.7/base $56 per gene, 1per pool 500×2×3=3,000; $84,000 @ 0.7/base, $8.85 per gene, 20phenotypes per pool, normalized abundance (e.g. by using only threevariable codons, two of which are degenerate: NNT, VAA, TGG)500×2=1,000; $28,000 @0.7/base $2.94 per gene, 20 phenotypes per pool,skewed abundance (this results in the presence of significant numbers oftruncated genes in the synthesized library).

The same physical oligo inventory used for the first round GAGGS is usedin the second round of GAGGS to synthesize a library which contains ˜95%of all possible combinations of any of two single aa changes. To have100% coverage (to include for combinations of mutations within +/−20 bpproximity, additional oligos are used. Where at least one mutation fromthe previous round has been identified as beneficial, coverage of allcombinations of new mutations within +/−20 bp of the beneficialmutations uses synthesis of no more than 42 new oligos). The cost ofsubsequent rounds of GAGGS grow only marginally, and linearly, whilediversity sampled in a recursive mode grows exponentially.

Example 4 GAGGS of Recombinogenic (Non-Mutagenic) Library Parented by aFamily of Genes (GAGGS Equivalent of Single Round of Family DNAShuffling)

Given sequence information for six fairly average (1.6 kb) size genes,each having six areas of homology with each of the other parental genes(six “heads” and “tails” for chimerizing each area of homology).

Relevant parameters include: the number of oligos and resulting cost tobuild 6 parental 1.6 kb genes (from 40 mer oligos, complete 20+20overlaps, by non-error-prone assembly PCR), the number of distinctpairwise crossovers between all matching homology areas, assuming 1crossover event per pairwise homology region), the number of allpossible chimeras using the crossovers, the theoretical library size,and the number of distinct oligos and cost to build all possiblechimeras. As above, 6 4 1.600:40 4 2=480 oligos, $0.7 4 40 4480=$13,440, i.e., $2,240 per gene built. N=180, calculated according tothe formula N=k ∞ m ∞ (m−1), where m=6 number of parents, and k=6 is thenumber of pairwise homology areas satisfying crossover conditions.X=˜5.315 ∞ 10⁹, calculated according to the formula:

$X = {\sum\limits_{n = 1}^{k}\left\{ {C{\begin{matrix}n \\k\end{matrix} \times m \times \left\lbrack {m \times \left( {m - 1} \right)} \right\rbrack^{n}}} \right\}}$

where X is the theoretical library size and n is number of crossovers ineach library entry (integer from 1 to k) 2 ∞ 180+480=840 oligos; $0.7 ∞40 ∞ 840=$23,520; $0.000048 per gene built. If only 10⁶ are screened,then the cost of oligos is $0.024 per gene built; if only 10⁵, then thecost of oligos is $0.24 per gene built, if 10⁴ are screened then thecost of oligos is $2.35 per gene built.

The cost of running multiple rounds of GAGGS is not additive, as most ofthe excess oligos from previous rounds can be reused in synthesis of thelater generation libraries. Even if only a small fraction of all genesbuilt is actually screened (e.g. 10⁴, with cost of oligos $2.35 per genebuilt), the oligo expenses are comparable with cost of assays on pergene-assay basis. In addition, industry wide oligo synthesis costs aredeclining.

Example 5 Stepwise GAGGS

This example provides a GAGGS family model stepwise protocol.

A family of genes/proteins (DNA or AA sequence) is selected. Allpossible pairwise alignments are made to identify pairwise homologyregions satisfying crossover operator conditions (length, % identity,stringency). Crossover points are selected, one per each of the pairwisehomology substrings, in the middle of each substring, or randomly, oraccording to an annealing-based probability model built on histograms ofcrossover probability ranks for every pair of parents. Oligos areselected for assembly PCR and synthesized. Genes/libraries are assembledfrom synthesized oligos. The libraries are screened/selected as setforth above.

Example 6 Subtilisin Family Model

Amino acid sequences were aligned (Codon usage can be optimized onretrotranslation for a preferred expression system, and number of oligosfor synthesis can be minimized). A Dot plot pairwise alignment of allpossible pairs of 7 parents was made (FIGS. 5, 6, 7). FIG. 5 is apercent similarity alignment for 7 parents. Amino acid sequences arealigned, with the leader peptide excluded. FIG. 6 is a dot-plotalignment of the sequences to identify regions of similarity. FIG. 7 isa clot plot showing pairwise crossover points in the alignment.

Pair 6 and 7 show 95% percent identity per each window of ≧7aa, whileall other pairs show 80% percent identity per each window of ≧7aa. Notethat the stringency of alignment (and subsequent representation ofcrossover between parents) can be manipulated individually for eachpair, so that low homology crossovers can be represented at the expenseof highly homologous parents. No structural biases or active site biaseswere incorporated in this model.

As an example GAGGS calculation for the subtilisin family model,assuming 7 parents, of about 400 amino acids, and 1200 bp each(including the leader) or about 275 amino acids and 825 bp for matureprotein, 7×825×2+about 500=12 kb of total sequence to be generated bygene synthesis. From 40 mers, with full overlap assembly (20+20 bpoverlaps), about 300 oligos are used.

For pairwise crossover oligos to build chimeras, based on alignmentresults, with one crossover per each homologous substring, there areabout 180 homologous substrings, with 170 in the coding region and 10 inthe leader region. With 260 mers per each crossover point, and 2head-tail sets for each pair of parents, about 360 additional oligosdedicated to build crossovers can be used. The total number of oligos isabout 660 (300 40 mers and 360 60 mers). At a total cost of oligos of$0.70 per base, the oligos would be about $23,520. The cost of reagentswould run about $0.07 per base, for a total cost of about $2,252dollars.

Example 7 Napthalene Dioxygenase

Napthalene dioxygenase is a non-heme reductive dioxygenase. There are atleast three closely related but catalytically distinct types ofNapthalene dioxygenases. FIG. 12 provides a schematic of a percentsimilarity plot for the three different Napthalene dioxygenase types,with the amino acid sequence for the ISP large subunit (which isresponsible for substrate specificity) being provided.

At a size of about 1,400 amino acids, there are 3×1,400 total basepairs=260 40 mer oligos for 20-X20 overlap gene synthesis. A plot of thesequence alignment reveals that there are 14+19+23=11260 mer highstringency oligos used in the recombination. The cost of oligos at $0.70per base would yield a cost of about $12,000 for synthesis, using about9 hours of synthesizer time to make the oligos. The estimated librarysize would be about 9.4×10⁹ chimeras.

Example 8 Single Parent GAGGS Calculation

As noted above, one aspect of the invention provides for single parentGAGGS. In these methods, polynucleotides having desired characteristicsare provided. This is accomplished by: (a) providing a parental sequencecharacter string encoding a polynucleotide or polypeptide; (b) providinga set of character strings of a pre-defined length that encodesingle-stranded oligonucleotide sequences comprising overlappingsequence fragments of an entire parental character string, and an entirepolynucleotide strand complementary to the parental character string(splitting the sequence of a parent into oligos suitable for assemblyPCR); (c) creating a set of derivatives of parental sequence comprisingvariants with all possible single point mutations, with, e.g., onemutation per variant string (defining all possible single pointmutations); (d) providing a set of overlapping character strings of apre-defined length that encode both strands of the parentaloligonucleotide sequence, and a set of overlapping character strings ofa pre-defined length that encode sequence areas including the mutations(oligos incorporating single point mutations, suitable for the sameassembly PCR scheme); (e) synthesizing sets of single-strandedoligonucleotides according to the step (c) (e.g., to build or rebuildthe parental sequence or a variant thereof e.g., incorporating singlepoint mutations during gene assembly); (f) assembling a library ofmutated genes in assembly PCR from the single-stranded oligonucleotides(pooling, partial pooling, or one per container).

For one gene per container approaches (or other approaches involvingphysically separating library components, e.g., in arrays), wild typeoligos are excluded at mutations; and (g) selecting or screening forrecombinant polynucleotides having evolved toward a desired property. Inan additional optional step (h), the method includes deconvolutingsequence of the mutated polynucleotides (i.e., determining which librarymember has a sequence of interest, and what that sequence is) havingevolved toward a desired property to determine beneficial mutations(when assembly PCR is one per container format, this is done bypositional sequence deconvolution, rather than actual sequencing, i.e.,the physical location of the components are adequate to provideknowledge of the sequence). In an optional additional step (i), themethod includes assembling a library of recombinant variants whichcombine some or all possible beneficial mutations in some or allpossible combinations, from single-stranded oligos by assembly PCR. Thisis performed from the same set of oligos; if some of the mutations arepositionally close (within any one oligo), then additional single strandoligos are made which incorporate combinations of mutations. An optionalstep (j) includes selecting or screening for recombinant polynucleotideshaving evolved further toward a desired property.

An example single parent GAGGS calculation, per 1 kb of sequencefollows.

Genome length: 1000 bp.

First round mutation rate: 1 amino acid/gene.

Number of oligonucleotides to build wild-type gene: 52 (40 mers, 20+20overlap synthesis scheme).

Number of oligonucleotide to provide for all possible single pointnon-terminating mutations (333 total possible): non degenerate oligos:13320. Partially degenerate 40 mers, one pg position per oligo: 1920.Fully degenerate oligos, 40 mers, one fg position per oligo: 666.

Error prone PCR assembly will also work, but sequence deconvolution isperformed, e.g., by sequencing before subsequent rounds.

The number of additional oligos to allow for construction of allpossible recombinations with beneficial mutations would use about 10% ofthe preceding number of oligos. However, about 95% of all possiblerecombinants having beneficial mutations can be made from the initialset produced above.

Example 9 A Process for Design of Crossover Oligonucleotides forSynthesis of Chimerical Polynucleotides

First, substrings are identified and selected in parental strings forapplying a crossover operator to from chimeric junctions. This isperformed by: a) identifying all or part of the pairwise homologyregions between all parental character strings, b) selecting all or partof the identified pairwise homology regions for indexing at least onecrossover point within each of the selected pairwise homology regions,c) selecting one or more of the pairwise non-homology regions forindexing at least one crossover point within each of the selectedpairwise non-homology regions (“c” is an optional step which can beomitted, and is also a step where structure-activity based elitism canbe applied), thereby providing a description of a set of positionallyand parent-indexed regions/areas (substrings) of parental characterstrings suitable for further selection of crossover points.

Secondly, further selection of crossover points within each of thesubstrings of the set of the substrings selected in step 1 above isperformed. The steps include: a) randomly selecting at least one of thecrossover points in each of the selected substrings, and/or b) selectingat least one of the crossover points in each of the selected substrings,using one or more of annealing-simulation-based models for determiningprobability of the crossover point selection within each of the selectedsubstrings and/or c) selecting one crossover point approximately in themiddle of each of the selected substrings, thereby creating a set ofpairwise crossover points, where each point is indexed to correspondingcharacter positions in each of the parental strings desired to from achimeric junction at that point.

Thirdly, optional codon usage adjustments are performed. Depending onmethods used to determine homology (strings encoding DNA or AA), theprocess can be varied. For example, if a DNA sequences was used: a)adjustment of codons for the selected expression system is performed forevery parental string, and b) adjustment of codons among parents can beperformed to standardize codon usage for every given aa at everycorresponding position. This process can significantly decrease totalnumber of distinct oligos for gene library synthesis, and may beparticularly beneficial for cases where AA homology is higher than DNAhomology, or with families of highly homologous genes (e.g. 80%+identical).

This option has to be exercised with caution, as it is in essence anexpression of an elitism mutation operator. Thus, one considers thebenefits of cutting the number and resulting costs of oligos vs.introduction of this bias, which can have undesirable consequences. Mosttypically, one uses codons which encode AA at a given position in amajority of parents.

If AA sequences are used: a) retrotranslate sequence to degenerate DNA;b) define degenerate nucleotides using position-by-position referencingto codon usage in original DNA (of majority of parents or ofcorresponding parent), and/or—exercise codon adjustments suitable forthe selected expression system where a physical assay will be performed.

This step can also be used to introduce any restriction sites withincoding parts of the genes, if any, for subsequentidentification/QA/deconvolution/manipulations of library entries. Allcrossover points identified in step 2 above (indexed to pairs ofparents) are correspondingly indexed to the adjusted DNA sequences.

Fourth, oligo arrangements are selected for a gene assembly scheme. Thisstep includes several decision steps:

Uniform 40-60 mer oligos are typically used (using longer oligos willresult in decrease of # of oligos to build parents, but uses additionaldedicated oligos for providing representation of closely positionedcrossovers/mutations.

Select whether Shorter/Longer Oligos are allowed (i.e., a Yes/Nodecision). A “Yes” decision cuts the total number of oligos for highhomology genes of different lengths with gaps (deletion/insertion), espfor 1-2aa.

Select the overlap length (typically 15-20 bases, which can besymmetrical or asymmetrical).

Select whether degenerate oligos are allowed (Yes/No). This is anotherpotent cost cutting feature and also a powerful means to obtainadditional sequence diversity. Partial degeneracy schemes and minimizeddegeneracy schemes are especially beneficial in building mutageniclibraries.

If software tools are used for these operations, several variations ofthe parameters are run to select maximum library complexity and minimalcost. Exercising complex assembly schemes using oligos of various lengthsignificantly complicates indexing processes and, subsequently, assemblyof the library in positionally encoded parallel or partial poolingformats. If this is done without sophisticated software, a simple anduniform scheme (e.g. all oligos 40 bases long with 20 bases overlap) canbe used.

Fifth, “convenience sequences” are designed in front and in the back ofthe parent strings. Ideally, it is the same set which will be built inevery library entry at the end. These include any restriction sites,primer sequences for assembled product identifications, RBS, leaderpeptides and other special or desirable features. In principle, theconvenience sequences can be defined at a later stage, and at thisstage, a “dummy” set of appropriate length can be used, e.g. a substringfrom an easily recognizable forbidden letters.

Sixth, an indexed matrix of oligo strings for building every parent iscreated, according to the selected scheme. An index of every oligoincludes: a parent identifier (parentID), indication of coding orcomplementary chain, and position numbers. Crossover-points aredetermined for indexed coding string of every parent with head and tailconvenience substrings. A complementary chain of every string isgenerated. Every coding string is selected according to the selectedassembly PCR scheme in step 4 above (e.g. in increments of 40 bp). Everycomplement string is split according to the same scheme (e.g. 40 bp with20 bp shift)

Seventh, an indexed matrix of oligos is created for every pairwisecrossover operation. First, all oligos which have pairwise crossovermarkers are determined. Second, all sets of all oligos which have thesame position and same pair of parents crossover markers (4 percrossover point) are determined. Third, every set of 4 oligo strings aretaken which have been labeled with the same crossover marker, andanother derivative set of 4 chimeric oligo strings comprising ofcharacters encoding 2 coding and 2 complement chains (e.g. with 20 bpshift in 40=20+20 scheme) are made. Two coding strings are possible,having a forward end sequence substring of one parent followed by thebackward end of the second parent after crossover point. Complementstrings are also designed in the same fashion, thereby obtaining anindexed complete inventory of strings encoding oligos suitable for genelibrary assembly by PCR.

This inventory can further be optionally refined by detecting allredundant oligos, counting them and deleting from inventory, accompaniedby the introduction of the count value to an “abundance=amount” field inthe index of each oligo string. This may be a very beneficial step forreducing total number of oligos for library synthesis, particularly inthe cases if parental sequences are highly homologous.

Example 10 Program Algorithm for Designing Oligonucleotides forSynthesis

The following is a program outline for designing oligonucleotides foruse in synthetic/recombination protocols.

Given an alignment of proteins and a codon bias table:

For each position in an alignment of proteins  find a set of minimallydegenerate codons that code for Amino Acids at this position using codonbias table  For each sequence in alignment   Add three letter codon(DNA) that codes for the amino acid at this position in this sequence toDNA version of sequence   !Gaps are represent by a special codon ~~~ foreach sequence of DNA created by above  !Note gaps are ignored in thisstep  For each window=rough oligo size   check end degeneracy   Try toincrease and decrease window length to minimize end degeneracy whilestaying within length bounds   add oligos given window bounds and allsequences   add oligos given reverse window bounds and all sequences foreach position in dnaseqs  for each sequence   If the current position isthe start of a gap    Add oligos given bounds which contains a minimumamount of sequence from current sequence 5′ of the gap and a minimumamount of sequence from current sequence 3′ of the gap    Repeat addoligos for reverse bounds add Oligos: given a list of sequences (DNA)and bounds  for each position in the bounds   get all unique bases atthis position from DNA sequences in the list   generate a base (ordegenerate base symbol) for this position  if total degenerate positionsis greater than user defined number   split sequence list in two, addOligos given sequence list one, add Oligos given sequence list two(recursive)  else add this oligo (set of bases for each position) tooligo list display all oligo in oligo list

Example 11 Crossover Point Selection

FIGS. 8-11 are schematics of various processes and process criteria foroligonucleotide selection for recombination between parental nucleicacids. FIG. 8, panel A shows a typical dot-plot alignment of two parentsand the increase in crossover probability that results in regions ofsimilarity. Panel B shows that crossovers can be selected based upon asimple logical/physical filter, i.e., the physical or virtual annealingtemperature of oligonucleotides, e.g., using a linear annealingtemperature. Panel C shows various more complex filters which varyannealing temperature to achieve specific crossovers, i.e., byappropriately controlling the physical or virtual annealing temperature.

FIG. 9 schematically represents the introduction of indexed crossoverpoints into the sequence of each of the aligned parents. In brief,sequences are aligned and the positional index of each crossover point(marker field) is represented schematically by a vertical identifiermark. The crossover point, for parents m and n, as represented in FIG.8, is represented by an identifier, a position number for parent m (ahead) and a position number for parent n (a tail). This process isrepeated for every parent in a data set, applying the oligonucleotidegridding operator (grid of positional indexes which indicate the startand end of every oligonucleotide in a PCR assembly operation) to each ofthe parents. FIG. 10 schematically represents the complete inventory ofoligonucleotide sequences to assemble all of the parents. The data setis simplified by identifying all pairs of oligonucleotide sequences withmatching pairwise crossover indexes, providing a sub inventory of oligoswith crossover markers. FIG. 11 provides a schematic for obtaining aninventory of sequences for chimeric oligonucleotides for each of theselected crossover points. In brief, two pairs of oligo sequences withmatching pairwise crossover indexes are selected (down arrow 1).Sequences of chimeric oligonucleotides are generated around the pairwisecrossover point (head-tail, tail-head, s or a chain) (down arrow 2). Ina “40=20+20” assembly scheme (where a 40mer has 20 residues from eachparent), only one oligo longer than 60 bp. is used for eachchimerization (two for each crossover point, regardless of relativepositions within each oligo). This empirical finding can be described byvariations of cut and join operations at the depicted S or A chains(e.g., as in FIG. 8), with the rule being reduced to a guidance table.In the chimeric oligos, the A or S chain and sequence head and tailsubfragments are defined using a selected subset of rules from theguidance table. Selection or rules from the guidance table can beautomated based upon comparison of relative positions of crossoverpoints in the oligos (boolean operations) (down arrow 3). This processis repeated for every set of oligos with identical crossover indexes toobtain an inventory of sequences for chimeric oligos for each of theselected crossover points.

Example 12 Synthetic Shuffling by Repeated Cycles of Melting, Annealingand Polymerization

The methods herein provide, inter alia, synthetic shuffling using, e.g.,a primerless PCR reaction to assemble a series of overlapping oligosdesigned to capture the diversity represented in a family of relatedsequences. For example, the process provides family shuffling, withoutneeding access to starting parental sequences—synthetic shufflingprovides a direct route from an in silico database to shuffled library.See also, “OLIGONUCLEOTIDE MEDIATED NUCLEIC ACID RECOMBINATION” byCrameri et al., Filed Jan. 18, 2000, U.S. Ser. No. PCT/US00/01203.Synthetic shuffling projects are not limited to natural diversity, andneed not result in equal representation of diversity, and diversity canoccur with any desired granularity of diversity (e.g., at the level ofsingle amino acids, or in blocks, depending on the sequences that areselected). Freedom of oligo sequence, length, and number; as well asfreedom to mix oligos in any desired combination and quantities underany desired assembly parameters allows great control over librarydesign.

One example is the synthetic shuffling of subtilisins, also describedsupra. In one example 16 overlapping 60-mers (8 top strand and 8 bottomstrand) with 20 bp of homology between ends of top and bottom strandoligos formed a backbone were designed to capture the majority ofnatural diversity represented in a 660 bp gene segment (also known asthe diversified region) of a family of 15 subtilisin sequences. Thediversity was largely captured by introducing degeneracies into theoligonucleotide backbone. Diversity that was not captured in thebackbone oligos was encoded in II additional 39- to 45-mers. In additionto capturing the diversity represented in the 15 subtilisin sequences,the backbone and spiking oligos optimized codon usage for Bacillussubtilis and maximized recombination. Oligos were mixed at equimolarconcentrations and assembled in a primerless PCR reaction. A full-lengthlibrary was rescued from the assembly by conventional PCR with primersannealing to construct ends. The library was designed such that itrecombines at the level of single amino acids and results in equalrepresentation of all possible amino acids in a particular position(assuming unbiased assembly and equal representation of nucleotides atdegenerate positions). This is in contrast to recombination by simplefragmentation and reassembly, which typically shuffles in blocks and canresult in biased representation of encoded amino acids, based onoccurrence of a particular amino acid in the starting parentalsequences.

Modifications can be made to the methods and materials as hereinbeforedescribed without departing from the spirit or scope of the invention asclaimed, and the invention can be put to a number of different uses,including:

The use of an integrated system to generate shuffled nucleic acidsand/or to test shuffled nucleic acids, including in an iterativeprocess.

An assay, kit or system utilizing a use of any one of the selectionstrategies, materials, components, methods or substrates hereinbeforedescribed. Kits will optionally additionally comprise instructions forperforming methods or assays, packaging materials, one or morecontainers which contain assay, device or system components, or thelike.

In an additional aspect, the present invention provides kits embodyingthe methods and apparatus herein. Kits of the invention optionallycomprise one or more of the following: (1) a shuffled component asdescribed herein; (2) instructions for practicing the methods describedherein, and/or for operating the selection procedure herein; (3) one ormore assay component; (4) a container for holding nucleic acids orenzymes, other nucleic acids, transgenic plants, animals, cells, or thelike, (5) packaging materials, and (6) software for performing any ofthe decision steps noted herein related to GAGGS.

In a further aspect, the present invention provides for the use of anycomponent or kit herein, for the practice of any method or assay herein,and/or for the use of any apparatus or kit to practice any assay ormethod herein.

The previous examples are illustrative and not limiting. One of skillwill recognize a variety of non-critical parameters which may be alteredto achieve essentially similar results. All parents, applications andpublications cited herein are incorporated by reference in theirentirety for all purposes.

1. A method of making a recombinant nucleic acid, the method comprising:providing a plurality of parental character strings corresponding to aplurality of nucleic acids, which character strings, when aligned formaximum identity, comprise at least one region of heterology; aligningthe character strings; defining a set of character string subsequences,which set of subsequences comprises subsequences of at least two of theplurality of parental character strings; providing a set ofoligonucleotides corresponding to the set of character stringsubsequences; annealing members of the set of oligonucleotides with oneanother in vitro; after annealing, elongating one or more members of theset of oligonucleotides with a polymerase, or ligating at least twomembers of the set of oligonucleotides with a ligase, thereby producingone or more recombinant nucleic acid; selecting the recombinant nucleicacid, or a polypeptide encoded by the recombinant nucleic acid for adesired trait or property, thereby providing a selected recombinantnucleic acid; and recombining the selected recombinant nucleic acid withone or more of (i) a nucleic acid homologous to a parental characterstring, and (ii) an oligonucleotide member from the set ofoligonucleotides.
 2. The method of claim 1, wherein each parentalcharacter string when aligned for maximum identity contains areas ofidentity and areas of heterology with any other parental characterstring.
 3. The method of claim 1, wherein at least one of the parentalcharacter strings is an evolutionary or artificial intermediate.
 4. Themethod of claim 1, wherein at least one of the parental characterstrings corresponds to a designed nucleic acid or designed polypeptide.5. The method of claim 4, wherein the designed nucleic acid representsan energy minimized design for an encoded polypeptide.
 6. The method ofclaim 1, further comprising applying one or more genetic operator to oneor more of the parental character strings, or to one or more of thecharacter string subsequences, wherein the genetic operator is selectedfrom the group consisting of: a mutation of the one or more parentalcharacter strings or one or more character string subsequences, amultiplication of the one or more parental character strings or one ormore character string subsequences, a fragmentation of the one or moreparental character strings or one or more character string subsequences,a crossover between any of the one or more parental character strings orone or more character string subsequences or an additional characterstring, a ligation of the one or more parental character strings or oneor more character string subsequences, an elitism calculation, acalculation of sequence homology or sequence similarity of alignedstrings, a recursive use of one or more genetic operator for evolutionof character strings, application of a randomness operator to the one ormore parental character strings or the one or more character stringsubsequences, a deletion mutation of the one or more parental characterstrings or one or more character string subsequences, an insertionmutation into the one or more parental character strings or one or moreof character string subsequences, subtraction of the of the one or moreparental character strings or one or more character string subsequenceswith an inactive sequence, selection of the of the one or more parentalcharacter strings or one or more character string subsequences with anactive sequence, and death of the one or more parental character stringsor one or more of character string subsequences.
 7. The method of claim1, further comprising generating a diplomat sequence, which diplomatsequence comprises an intermediate level of sequence similarity betweentwo or more additional members of the plurality of parental characterstrings wherein the set of oligonucleotides comprises or encodessubsequences of the diplomat sequence.
 8. The method of claim 1, furthercomprising selecting one or more cross-over sites between two or more ofthe parental character strings and wherein the set of oligonucleotidescomprises one or more bridging oligonucleotides.
 9. The method of claim8, wherein the two or more parental sequences are less than 50% similar.10. The method of claim 8, further comprising determining a sequence forone or more putative recombinant nucleic acids or polypeptides resultingfrom in silico recombination of the two or more parental characterstrings at the cross-over sites, and performing one or more in silicosimulations of activity for one or more of the putative recombinantnucleic acid or polypeptide.
 11. The method of claim 10, furthercomprising synthesizing the putative recombinant nucleic acid byproviding fragments of the two or more parental nucleic acids and atleast one of the corresponding bridge oligonucleotides, hybridizing thefragments and the bridge oligonucleotides and elongating the hybridizedfragments with a polymerase or a ligase.
 12. The method of claim 1,wherein the set of oligonucleotides comprise a plurality of overlappingoligonucleotides.
 13. The method of claim 1, wherein the set ofcharacter string subsequences is defined by subdividing at least two ofthe plurality of parental character strings into segments of a selectedlength.
 14. The method of claim 1, wherein aligning the characterstrings is performed in a digital computer or in a network-based system.15. The method of claim 1, wherein providing the set of oligonucleotidescomprises synthesizing a set of single-stranded oligonucleotidescorresponding to the set of character string subsequences.
 16. Themethod of claim 1, wherein the set of oligonucleotides is provided bysynthesizing the oligonucleotides to comprise one or more modifiedparental character string subsequence, which subsequence comprises oneor more of: a parental character string subsequence modified by one ormore replacement of one or more character of the parental characterstring subsequence with one or more different character; a parentalcharacter string subsequence modified by one or more deletion orinsertion of one or more characters of the parental character stringsubsequence; a parental character string subsequence modified byinclusion of a degenerate sequence character at one or more randomly ornon-randomly selected positions; a parental character string subsequencemodified by inclusion of a character string from a different characterstring from a second parental character string subsequence at one ormore position; a parental character string subsequence which is biasedbased upon its frequency in a selected library of nucleic acids; and, aparental character string subsequence which comprises, or encodes apolypeptide that comprises, one or more sequence motif, which sequencemotif is artificially included in the subsequence.
 17. The method ofclaim 16, wherein the sequence motif comprises an N-linked glycosylationsequence, an O-linked glycosylation sequence, a protease sensitivesequence, a collagenase sensitive sequence, a Rho-dependenttranscriptional termination sequence, an RNA secondary structuresequence that affects the efficiency of transcription, an RNA secondarystructure sequence that affects the efficiency of translation, atranscriptional enhancer sequence, a transcriptional promoter sequence,or a transcriptional silencing sequence.
 18. The method of claim 1,wherein the oligonucleotide set contains one or more altered ordegenerate positions as compared to the corresponding subsequence of oneor more parental character string.
 19. The method of claim 1, whereinselecting the recombinant nucleic acid is based upon its hybridizationto a selected nucleic acid or to a set of selected nucleic acids. 20.The method of claim 1, wherein the character string subsequences fromthe at least two parental character strings are separated by a crossoverpoint.
 21. The method of claim 20, wherein the crossover point isselected by aligning at least one substring of each of at least two ofthe parental character strings to display pairwise identity between thesubstrings, and selecting a point within the aligned sequence as thecrossover point.
 22. The method of claim 20, wherein the crossover pointis selected randomly.
 23. The method of claim 20, wherein the crossoverpoint is selected non randomly.
 24. The method of claim 20, wherein thecrossover point is selected non randomly by selecting a crossover pointapproximately in the middle of one or more identified pairwise identityregion.
 25. The method of claim 1, wherein providing the set ofoligonucleotides comprises providing one or more oligonucleotide memberof the set of oligonucleotides at a concentration which is higher thanat least one or more additional oligonucleotide member of the set ofoligonucleotides.
 26. The method of claim 1, further comprisingdenaturing the selected recombinant nucleic acid, and contacting therecombinant nucleic acid with at least one additional nucleic acid fromthe oligonucleotide set.
 27. The method of claim 1, further comprisingdenaturing the selected recombinant nucleic acid, and contacting the oneor more recombinant nucleic acid with at least one additional nucleicacid produced by cleavage of at least one parental nucleic acid.
 28. Themethod of claim 1, further comprising denaturing the selectedrecombinant nucleic acid, and contacting the recombinant nucleic acidwith at least one additional nucleic acid produced by cleavage of aparental nucleic acid, which parental nucleic acid is cleaved by one ormore of: chemical cleavage, cleavage with a DNAse, and cleavage with arestriction endonuclease.
 29. The method of claim 1, wherein at leastone parental nucleic acid encodes a protein selected from:erythropoietin (EPO), insulin, a peptide hormone, a cytokine, epidermalgrowth factor, fibroblast growth factor, hepatocyte growth factor,insulin-like growth factor, an interferon, an interleukin, akeratinocyte growth factor, a leukemia inhibitory factor, oncostatin M,platelet derived erythroid colony stimulating factor (PD-ECSF),Platelet-derived growth factor (PDGF), pleiotropin, stem cell factor(SCF), c-kit ligand, vascular endothelial growth factor (VEGF),granulocyte-colony stimulating factor (G-CSF), an oncogene product, atumor suppressor, a steroid hormone receptor, a plant hormone, a diseaseresistance gene, an herbicide resistance gene product, a bacterial geneproduct, a monooxygenase, a protease, a nuclease, and a lipase.
 30. Themethod of claim 1, wherein the set of oligonucleotides comprises one ormore oligonucleotide member of between about 20 and about 60 nucleotidesin length.
 31. The method of claim 1, wherein the selected recombinantnucleic acid is selected for an in vivo selection assay or a parallelsolid phase assay.
 32. The method of claim 1, wherein the selectedrecombinant nucleic acid is selected for an in vitro selection assay.33. The method of claim 1, further comprising deconvolution of therecombinant nucleic acid.
 34. The method of claim 1, further comprisingsequencing or cloning the selected recombinant nucleic acid.
 35. Themethod of claim 1, wherein the set of oligonucleotides is synthesized invitro by assembly PCR.
 36. The method of claim 1, wherein the set ofoligonucleotides is synthesized in vitro by error-prone assembly PCR.37. The method of claim 1, wherein one or more of the parental characterstrings comprises a diplomat sequence.